Modified clostridial toxins with enhanced translocation capabilitiy

ABSTRACT

The specification discloses modified Clostridial toxins comprising a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain, a translocation facilitating domain and an enhanced targeting domain; polynucleotide molecules encoding such modified Clostridial toxins; and method of producing such modified Clostridial toxins.

This application is a divisional and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 11/776,043, filed Jul.11, 2007, which claims priority pursuant to 35 U.S.C. §119(e) to U.S.Provisional Patent Application Ser. No. 60/807,063 filed Jul. 11, 2006,both incorporated entirely by reference.

All patents and publications cited in this application are herebyincorporated by reference in their entirety.

The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins(BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, andTetanus neurotoxin (TeNT), to inhibit neuronal transmission are beingexploited in a wide variety of therapeutic and cosmetic applications,see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OFBOTULINUM TOXIN (Slack, Inc., 2004). Clostridial toxins commerciallyavailable as pharmaceutical compositions include, BoNT/A preparations,such as, e.g., BOTOX® (Allergan, Inc., Irvine, Calif.),Dysport®/Reloxin®, (Beaufour Ipsen, Porton Down, England), Linurase®(Prollenium, Inc., Ontario, Canada), Neuronox® (Medy-Tox, Inc.,Ochang-myeon, South Korea) BTX-A (Lanzhou Institute Biological Products,China) and Xeomin® (Merz Pharmaceuticals, GmbH., Frankfurt, Germany);and BoNT/B preparations, such as, e.g., MyoBloc™/NeuroBloc™ (ElanPharmaceuticals, San Francisco, Calif.). As an example, BOTOX® iscurrently approved in one or more countries for the followingindications: achalasia, adult spasticity, anal fissure, back pain,blepharospasm, bruxism, cervical dystonia, essential tremor, glabellarlines or hyperkinetic facial lines, headache, hemifacial spasm,hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy,multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodicdysphonia, strabismus and VII nerve disorder.

Clostridial toxin therapies are successfully used for many indications.Generally, administration of a Clostridial toxin treatment is welltolerated. However, toxin administration in some applications can bechallenging because of the larger doses required to achieve a beneficialeffect. First, larger doses can increase the likelihood that the toxinmay move through the interstitial fluids and the circulatory systems,such as, e.g., the cardiovascular system and the lymphatic system, ofthe body, resulting in the undesirable dispersal of the toxin to areasnot targeted for toxin treatment. Such dispersal can lead to undesirableside effects, such as, e.g., inhibition of neurotransmitter release inneurons not targeted for treatment or paralysis of a muscle not targetedfor treatment. For example, a patient administered a therapeuticallyeffective amount of a BoNT/A treatment into the neck muscles fortorticollis may develop dysphagia because of dispersal of the toxin intothe oropharynx. Thus, there remains a need for improved Clostridialtoxins that are effective at the site of treatment, but have negligibleto minimal effects in areas not targeted for a toxin treatment.

Second, larger doses of a Clostridial toxin treatment may elicit anantibody response against the toxin. While a potent and effectivetreatment, the inhibition of neurotransmitter release and the resultingneuromuscular paralysis elicited by Clostridial toxin therapies is notpermanent. The reversible nature of these paralytic effects requiresperiodic treatments in order to maintain the therapeutic benefits fromthis toxin. As a consequence of this repeated exposure, an immuneresponse against a Clostridial toxin can occur in some patients whichreduce or completely prevent the individual's responsiveness to furthertreatments, see, e.g., Joseph Jankovic, Botulinum toxin: ClinicalImplications of Antigenicity and Immunoresistance, (SCIENTIFIC ANDTHERAPEUTIC ASPECTS OF BOTULINUM TOXIN, 409-415, Mitchell F. Brin etal., eds., Lippincott Williams & Wilkins, 2002); Dirk Dressler, ClinicalPresentation and Management of Antibody-induced Failure of BotulinumToxin Therapy, 19(Suppl. 8) MOV. DISORD. S92-S100 (2004); M. ZouhairAtassi, Basic Immunological Aspects of Botulinum Toxin Therapy,19(Suppl. 8) MOV. DISORD. S68-S84, (2004). Thus, there remains a needfor improved Clostridial toxins that maintain effective therapeuticbenefits, but have reduced ability to evoke an immunogenic responseagainst itself.

The growing clinical, therapeutic and cosmetic use of Clostridial toxinsin therapies requiring larger doses necessitates the pharmaceuticalindustry to develop modified Clostridial toxins that are effective atthe target site of the application, but reduce or prevent theundesirable side-effects associated with the dispersal of the toxins tounwanted locations and reduce or prevent an unwanted immunogenicresponse. One approach involves modifying a Clostridial toxin so thatthe modified toxin has an enhanced cell binding activity for anaturally-occurring Clostridial toxin receptor. This enhanced bindingactivity is achieved by modifying the endogenous targeting domain of anaturally-occurring Clostridial toxin in order to enhance a cell bindingactivity of the toxin for its naturally-occurring receptor. Suchmodifications to a targeting domain result in, e.g., a enhanced cellbinding activity that increases binding affinity for an endogenousClostridial toxin receptor present on a naturally-occurring Clostridialtoxin target cell; an enhanced cell binding activity that increasesbinding specificity for a subgroup of endogenous Clostridial toxinreceptors present on a naturally-occurring Clostridial toxin targetcell; or an enhanced cell binding activity that increases both bindingaffinity and binding specificity. An added advantage is achieved whenthe targeting domain is derived from a human targeting domainpolypeptide as these polypeptides as less likely to elicit animmunogenic response in a patient. A modified Clostridial toxin with anenhanced binding activity can bind to a receptor, translocate into thecytoplasm, and exert its proteolytic effect on the SNARE complex of theClostridial toxin target cell.

Non-limiting examples of modified Clostridial toxins an enhanced cellbinding activity for a naturally-occurring Clostridial toxin receptorare described in, e.g., Lance E. Steward, et al., Modified ClostridialToxins with Enhanced Targeting Capabilities For Endogenous ClostridialToxin Receptors, International Patent Application No. 2006/008956 (Mar.14, 2006). This enhanced binding activity for a naturally occurringClostridial toxin receptor allows for lower effective doses of amodified Clostridial toxin to be administered to an individual becausemore toxins will be delivered to the target cell. Thus, modifiedClostridial toxins with an enhanced cell binding activity for aClostridial toxin receptor will reduce the undesirable dispersal of thetoxin to areas not targeted for treatment, thereby reducing orpreventing the undesirable side-effects associated with diffusion of aClostridial toxin to an unwanted location.

The present invention provides novel modified Clostridial toxins thatreduce or prevent unwanted side-effects associated with toxin dispersalinto non-targeted areas. These modified Clostridial toxins comprise, inpart, a translocation facilitator domain that enhances the process bywhich a light chain from a modified toxin translocates into thecytoplasm of a target cell and enzymatically modify its target SNAREsubstrate. Thus modified Clostridial toxins with enhanced cell bindingactivity for its naturally-occurring receptor, such as, e.g., thosedisclosed in Steward, supra, (2006), comprising a translocationfacilitator domain disclosed in the present specification, exhibitincreased potency at the Clostridial toxin target cell and a concomitantreduction of unwanted side-effects associated with toxin dispersal intonon-targeted areas. These and related advantages are useful for variousclinical, therapeutic and cosmetic applications, such as, e.g., thetreatment of neuromuscular disorders, neuropathic disorders, eyedisorders, pain, muscle injuries, headache, cardiovascular diseases,neuropsychiatric disorders, endocrine disorders, cancers, otic disordersand hyperkinetic facial lines, as well as, other disorders whereadministration of a modified Clostridial toxin with enhanced cellbinding activity for a Clostridial toxin receptor to an individual canproduce a beneficial effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a schematic of the current paradigm ofneurotransmitter release and Clostridial toxin intoxication in a centraland peripheral neuron. FIG. 1A shows a schematic for theneurotransmitter release mechanism of a central and peripheral neuron.The release process can be described as comprising two steps: 1) vesicledocking, where the vesicle-bound SNARE protein of a vesicle containingneurotransmitter molecules associates with the membrane-bound SNAREproteins located at the plasma membrane; and 2) neurotransmitterrelease, where the vesicle fuses with the plasma membrane and theneurotransmitter molecules are exocytosed. FIG. 1B shows a schematic ofthe intoxication mechanism for tetanus and botulinum toxin activity in acentral and peripheral neuron. This intoxication process can bedescribed as comprising four steps: 1) receptor binding, where aClostridial toxin binds to a Clostridial receptor and initiates theintoxication process; 2) complex internalization, where after toxinbinding, a vesicle containing the toxin/receptor complex is endocytosedinto the cell; 3) light chain translocation, where multiple eventsresult in the release of the active light chain into the cytoplasm; and4) enzymatic target modification, where the active light chain ofClostridial toxin proteolytically cleaves its target SNARE substrate,such as, e.g., SNAP-25, VAMP or Syntaxin, thereby preventing vesicledocking and neurotransmitter release.

FIG. 2 shows the domain organization of naturally-occurring Clostridialtoxins. The single chain form depicts the amino to carboxyl linearorganization comprising an enzymatic domain, a translocation domain, aH_(CN) translocation facilitating domain and a H_(CC) targeting domain.The di-chain loop region located between the translocation and enzymaticdomains is depicted by the double SS bracket. This region comprises anendogenous di-chain loop protease cleavage site that upon proteolyticcleavage with a naturally-occurring protease, such as, e.g., anendogenous Clostridial toxin protease or a naturally-occurring proteaseproduced in the environment, converts the single chain form of the toxininto the di-chain form. As depicted above the single-chain form, theH_(CC) targeting domain comprises the β-trefoil domain which comprisesin an amino to carboxyl linear organization of an α-fold, a β4/β5hairpin turn, a β-fold, a β8/β9 hairpin turn and a γ-fold.

FIG. 3 shows a ribbon diagram of BoNT/A illustrating the modularthree-dimensional structure of the light chain (LC) comprising theenzymatic domain, the heavy chain H_(N) domain comprising thetranslocation domain, the heavy chain H_(CN) domain comprising thetranslocation facilitating domain and the heavy chain H_(CC) domaincomprising the targeting domain.

FIGS. 4A and 4B show modified Clostridial toxins with an enhancedtranslocation capability and an enhanced targeting activity located atthe amino terminus of the modified toxin. FIG. 4A depicts the singlepolypeptide form of a modified Clostridial toxin with an amino tocarboxyl linear organization comprising an enhanced targeting domain, atranslocation domain, a translocation facilitating domain and anenzymatic domain, with the di-chain loop region depicted by the doubleSS bracket. A proteolytic cleavage site (P) within a di-chain loopregion is located between the translocation facilitating and enzymaticdomains. Upon proteolytic cleavage with a P protease, the single chainform of the toxin is converted to the di-chain form. The P protease sitecan be a Clostridial toxin endogenous protease cleavage site or anon-Clostridial toxin exogenous protease cleavage site. Spacers can beplaced between the targeting and translocation domains, thetranslocation and translocation facilitating domains, translocationfacilitating and enzymatic domains or any combination thereof. FIG. 4Bdepicts the single polypeptide form of a modified Clostridial toxin withan amino to carboxyl linear organization comprising an enhancedtargeting domain, an enzymatic domain, a translocation domain and atranslocation facilitating domain, with the di-chain loop regiondepicted by the double SS bracket. A proteolytic cleavage site (P)within a di-chain loop region is located between the enzymatic andtranslocation domains. Upon proteolytic cleavage with a P protease, thesingle chain form of the toxin is converted to the di-chain form. The Pprotease site can be a Clostridial toxin endogenous protease cleavagesite or a non-Clostridial toxin exogenous protease cleavage site.Spacers can be placed between the targeting and enzymatic domains, theenzymatic and translocation domains, translocation and translocationfacilitating domains or any combination thereof.

FIGS. 5A and 5B show modified Clostridial toxins with an enhancedtranslocation capability and an enhanced targeting activity locatedbetween the two other domains. FIG. 5A depicts the single polypeptideform of a modified Clostridial toxin with an amino to carboxyl linearorganization comprising an enzymatic domain, an enhanced targetingdomain, a translocation domain and a translocation facilitating domain,with the di-chain loop region depicted by the double SS bracket. Aproteolytic cleavage site (P) within a di-chain loop region is locatedbetween the enzymatic and targeting domains. Upon proteolytic cleavagewith a P protease, the single chain form of the toxin is converted tothe di-chain form. The P protease site can be a Clostridial toxinendogenous protease cleavage site or a non-Clostridial toxin exogenousprotease cleavage site. Spacers can be placed between the enzymatic andtargeting domains, the targeting and translocation domains, thetranslocation and translocation facilitating domains or any combinationthereof. FIG. 5B depicts the single polypeptide form of a modifiedClostridial toxin with an amino to carboxyl linear organizationcomprising a translocation domain, a translocation facilitating domain,an enhanced targeting domain and an enzymatic domain, with the di-chainloop region depicted by the double SS bracket. A proteolytic cleavagesite (P) within a di-chain loop region is located between thetranslocation facilitating and targeting domains. Upon proteolyticcleavage with a P protease, the single chain form of the toxin isconverted to the di-chain form. The P protease site can be a Clostridialtoxin endogenous protease cleavage site or a non-Clostridial toxinexogenous protease cleavage site. Spacers can be placed between thetranslocation and translocation facilitating domains, the translocationfacilitating and targeting domains, the targeting and enzymatic domainsor any combination thereof.

FIGS. 6A and 6B show modified Clostridial toxins with an enhancedtranslocation capability and an enhanced targeting activity located atthe carboxyl terminus of the modified toxin. FIG. 6A depicts the singlepolypeptide form of a modified Clostridial toxin with an amino tocarboxyl linear organization comprising an enzymatic domain, atranslocation domain, a translocation facilitating domain and anenhanced targeting domain, with the di-chain loop region depicted by thedouble SS bracket. A proteolytic cleavage site (P) within a di-chainloop region is located between the enzymatic and translocation domains.Upon proteolytic cleavage with a P protease, the single chain form ofthe toxin is converted to the di-chain form. The P protease site can bea Clostridial toxin endogenous protease cleavage site or anon-Clostridial toxin exogenous protease cleavage site. Spacers can beplaced between the enzymatic and translocation domains, thetranslocation and translocation facilitating domains, the translocationfacilitating and targeting domains or any combination thereof. FIG. 6Bdepicts the single polypeptide form of a modified Clostridial toxin withan amino to carboxyl linear organization comprising a translocationdomain, a translocation facilitating domain, an enzymatic domain and anenhanced targeting domain, with the di-chain loop region depicted by thedouble SS bracket. A proteolytic cleavage site (P) within a di-chainloop region is located between the translocation facilitating andenzymatic domains. Upon proteolytic cleavage with a P protease, thesingle chain form of the toxin is converted to the di-chain form. The Pprotease site can be a Clostridial toxin endogenous protease cleavagesite or a non-Clostridial toxin exogenous protease cleavage site.Spacers can be placed between the translocation and translocationfacilitating domains, the translocation facilitating and enzymaticdomains, the enzymatic and targeting domains or any combination thereof.

FIGS. 7A and 7B show a ribbon diagram of BoNT/A illustrating theboundary regions of the H_(CN) domain comprising the translocationfacilitating domain. FIG. 7A depicts the amino-terminal boundary region.FIG. 7B depicts the carboxyl-terminal boundary region.

DETAILED DESCRIPTION

Clostridia toxins produced by Clostridium botulinum, Clostridium tetani,Clostridium baratii and Clostridium butyricum are the most widely usedin therapeutic and cosmetic treatments of humans and other mammals.Strains of C. botulinum produce seven antigenically-distinct types ofBotulinum toxins (BoNTs), which have been identified by investigatingbotulism outbreaks in man (BoNT/A, /B, /E and /F), animals (BoNT/C1 and/D), or isolated from soil (BoNT/G). While all seven botulinum toxins(BoNT) serotypes have similar structure and pharmacological properties,each also displays heterogeneous bacteriological characteristics. Incontrast, tetanus toxin (TeNT) is produced by a uniform group of C.tetani. Two other species of Clostridia, C. baratii and C. butyricum,also produce toxins similar to BoNT/F and BoNT/E, respectively.

Clostridia toxins possess approximately 35% amino acid identity witheach other and share the same functional domain organization and overallstructural architecture. Clostridial toxins are each translated as asingle chain polypeptide of approximately 150 kDa that is subsequentlycleaved by proteolytic scission within a disulfide loop by anaturally-occurring protease, such as, e.g., an endogenous Clostridialtoxin protease or a naturally-occurring protease produced in theenvironment (FIG. 2). This posttranslational processing yields adi-chain molecule comprising an approximately 50 kDa light chain (LC)and an approximately 100 kDa heavy chain (HC) held together by a singledisulfide bond and noncovalent interactions. It is widely held that themature di-chain molecule comprises three functionally distinctdomains: 1) an enzymatic domain located in the LC that includes ametalloprotease region containing a zinc-dependent endopeptidaseactivity which specifically targets core components of theneurotransmitter release apparatus (Table 1); 2) a translocation domaincontained within the amino-terminal half of the heavy chain (H_(N)domain) that facilitates release of the LC from intracellular vesiclesinto the cytoplasm of the target cell (Table 1); and 3) a binding domainfound within the carboxyl-terminal half of the heavy chain (H_(C)domain) that determines the binding activity and binding specificity ofthe toxin to the receptor complex located at the surface of the targetcell (Table 1), see, e.g., Kathryn Turton et al., Botulinum and TetanusNeurotoxins: Structure, Function and Therapeutic Utility, 27(11) TrendsBiochem. Sci. 552-558. (2002); John A. Chaddock and P. M. H. Marks,Clostridial Neurotoxins: Structure-Function Led Design of NewTherapeutics, 63(5) Cell. Mol. Life. Sci. 540-551 (2006); and KeithFoster et al., Re-engineering the Target Specificity of ClostridialNeurotoxins—A Route To Novel Therapeutics, 9(2-3) Neurotox Res. 101-107(2006).

TABLE 1 Clostridial Toxin Reference Sequences and Regions SEQ ID H_(C)Toxin NO: LC H_(N) H_(CN) H_(CC) BoNT/A 1 M1-K448 A449-I873 I874-P1110Y1111-L1296 BoNT/B 2 M1-K441 A442-I860 L861-E1097 Y1098-E1291 BoNT/C1 3M1-K449 T450-I868 N869-E1111 Y1112-E1291 BoNT/D 4 M1-R445 D446-I864N865-E1098 Y1099-E1276 BoNT/E 5 M1-R422 K423-I847 K848-E1085 Y1086-K1252BoNT/F 6 M1-K439 A440-I866 K867-K1105 Y1106-E1274 BoNT/G 7 M1-K446S447-I865 S866-Q1105 Y1106-E1297 TeNT 8 M1-A457 S458- K882-E1127Y1128-D1315 L881

The binding, translocation and enzymatic activities of a Clostridialtoxin are all necessary to execute the overall cellular intoxicationmechanism whereby Clostridial toxins enter a neuron and inhibitneurotransmitter release is similar, regardless of serotype or subtype.The current paradigm describes the intoxication mechanism as comprisingat least four steps: 1) receptor binding, 2) complex internalization, 3)light chain translocation, and 4) enzymatic target modification (seeFIG. 1). The process is initiated when the H_(C) domain of a Clostridialtoxin binds to a toxin-specific receptor located on the plasma membranesurface of a target cell. The binding specificity of a receptor complexis thought to be achieved, in part, by specific combinations ofgangliosides and protein receptors that appear to distinctly compriseeach Clostridial toxin receptor complex. Once bound, the toxin/receptorcomplexes are internalized by endocytosis and the internalized vesiclesare sorted to specific intracellular routes. The translocation step, nowthought to be mediated by the H_(N) domain and further facilitated bythe H_(CN) domain, appears to be triggered by the acidification of thevesicle compartment. This process seems to initiate two importantpH-dependent structural rearrangements that increase hydrophobicity andpromote separation of the light chain from the heavy chain of the toxin.Once activated, light chain endopeptidase of the toxin is released fromthe intracellular vesicle into the cytosol where it appears tospecifically target one of three known core components of theneurotransmitter release apparatus. These core proteins,vesicle-associated membrane protein (VAMP)/synaptobrevin,synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, arenecessary for synaptic vesicle docking and fusion at the nerve terminaland constitute members of the soluble N-ethylmaleimide-sensitivefactor-attachment protein-receptor (SNARE) family. BoNT/A and BoNT/Ecleave SNAP-25 in the carboxyl-terminal region, releasing a nine ortwenty-six amino acid segment, respectively, and BoNT/C1 also cleavesSNAP-25 near the carboxyl-terminus. The botulinum serotypes BoNT/B,BoNT/D, BoNT/F and BoNT/G, and tetanus toxin, act on the conservedcentral portion of VAMP, and release the amino-terminal portion of VAMPinto the cytosol. BoNT/C1 cleaves syntaxin at a single site near thecytosolic membrane surface. The selective proteolysis of synaptic SNAREsaccounts for the block of neurotransmitter release caused by Clostridialtoxins in vivo. The SNARE protein targets of Clostridial toxins arecommon to exocytosis in a variety of non-neuronal types; in these cells,as in neurons, light chain peptidase activity inhibits exocytosis, see,e.g., Yann Humeau et al., How Botulinum and Tetanus Neurotoxins BlockNeurotransmitter Release, 82(5) Biochimie. 427-446 (2000); and GiovannaLalli et al., The Journey of Tetanus and Botulinum Neurotoxins inNeurons, 11(9) Trends Microbiol. 431-437, (2003).

The three-dimensional crystal structures of BoNT/A, BoNT/B and the H_(C)domain of TeNT indicate that the three functional domains of Clostridialneurotoxins are structurally distinct. The HEXXH consensus motif of thelight chain forms the tetrahedral zinc binding pocket of the catalyticsite located in a deep cleft on the protein surface that is accessibleby a channel. The structure of the H_(N) and H_(C) domains consistsprimarily of β-sheet topologies that are linked by a single α-helix. Thecylindrical-shaped H_(N) domain comprises two long amphipathic α-helicesthat resemble the coiled-coil motif found in some viral proteins. TheH_(N) domain also forms a long unstructured loop called the‘translocation belt,’ which wraps around a large negatively chargedcleft of the light chain that blocks access of the zinc atom to thecatalytic-binding pocket of active site. The H_(C) domain comprises twodistinct structural features of roughly equal size that indicatefunction. The first, designated the H_(CN) domain, is located in theamino half of the H_(C) domain. The H_(CN) domain forms a β-barrel,jelly-roll fold. The H_(CC) domain is the second domain that comprisesthe H_(C) domain. This carboxyl-terminal domain comprises a modifiedβ-trefoil domain which forms three distinct carbohydrate binding regionsthat resembles the carbohydrate binding moiety found in manysugar-binding proteins, such as, e.g., serum amyloid P, sialidase,cryia, insecticidal δ-endotoxin and lectins. Biochemical studiesindicate that the β-trefoil domain structure of the H_(CC) domainappears to mediate the binding to specific carbohydrate containingcomponents of the Clostridial toxin receptor on the cell surface, see,e.g., Krzysztof Ginalski et al., Structure-based Sequence Alignment forthe Beta-Trefoil Subdomain of the Clostridial Neurotoxin Family ProvidesResidue Level Information About the Putative Ganglioside Binding Site,482(1-2) FEBS Lett. 119-124 (2000). The H_(C) domain tilts away from theH_(N) domain exposing the surface loops and making them accessible forbinding. No contacts occur between the light chain and the H_(C) domain.

We know that only the H_(CC) domain participates in receptor bindingbecause the β-trefoil domains are restricted to this domain. Proteinscontaining the structural β-trefoil domain represents a diverse group ofproteins organized into at least eight superfamilies including thecytokines, MIR domain proteins, Ricin B-like lectins, agglutinins,Soybean trypsin inhibitor like proteins, Actin-crosslinking proteins,LAG-1 proteins and AbfB domain proteins, see, e.g., C. A. Orengo et al.,Protein Superfamilies and Domain Superfolds, 372 Nature 631-634 (1994);and Alexey G. Murzin et al., SCOP: A Structural Classification ofProteins Database for the Investigation of Sequences and Structures,247(4) J. Mol. Biol. 536-540 (1995). While having diverse cellularroles, members of these superfamilies mechanistically function viaprotein-protein associations through the β-trefoil domain. Of particularinterest is the fact that many of these members are specificallyinvolved in receptor interactions, including, e.g., the cytokinesuperfamily members Fibroblast Growth Factors (FGFs) and theInterleukin-1s (IL-1s); the Ricin B-like lectins; the agglutinins; andSTI-like members the Kunitz inhibitors and Clostridium neurotoxins. Thatonly the H_(CC) domain alone mediates the cell binding step ofintoxication is further supported by the finding that mutations thatdisrupt the receptor binding activity of Clostridial toxins have beenconfined to the H_(CC) domain, see, e.g., Andreas Rummel et al., The H_(CC)-Domain of Botulinum Neurotoxins A and B Exhibits a SingularGanglioside Binding Site Displaying Serotype Specific CarbohydrateInteraction, 51(3) Mol. Microbiol. 631-643 (2004).

Because the H_(CC) domain appears not only necessary, but sufficient forselective binding of a Clostridial toxin to its receptor, we havededuced that the primary function of the H_(CN) domain of Clostridialtoxins is involved in the translocation step of the intoxicationprocess, and not in the cell binding step, because the lack of H_(CN)domain appears to reduce intoxication efficiency. For example, amodified BoNT/A comprising a Substance P targeting domain wasinefficient in intoxicating its corresponding target cells. In thismodified toxin, the entire BoNT/A H_(C) domain, comprising both theBoNT/A H_(CC) domain and the BoNT/A H_(CN) domain, was replaced by theSubstance P targeting domain. Likewise, we have determined that severalother modified Clostridial toxins that have replaced the entire BoNT/AH_(C) domain with an exogenous targeting domain have exhibited reducedintoxication capabilities. Thus, the H_(CN) domain possess atranslocation facilitating function because 1) H_(CC) domain primarilymediates the receptor binding step of the intoxication process; 2)modified Clostridial toxins lacking the H_(CN) domain exhibit a reducedability to translocate into the cytoplasm as evident by such modifiedtoxins exhibiting decreased proteolysis of their SNARE substrates; and3) the LC domain mediates the enzymatic activity of the toxin. While theexact translocation facilitating mechanism of the H_(CN) domain iscurrently not understood, the H_(CN) domain may 1) participate in theformation of an endosomal pore; 2) mediate the insertion of the poreinto a vesicle membrane; 3) assist in the delivery of LC across theendosomal membrane and/or 4) serve as a structural scaffold or spacerthat facilitates the appropriate orientation a the targeting domain inrelationship to the translocation domain. In this last point, the H_(CN)domain would serve to orient the translocation domain to facilitate theproper presentation of the translocation domain for insertion into themembrane following binding of the ligand by the receptor. This novelrole of the H_(CN) domain in the translocation step is contrary to thewidely accepted view that the Clostridial toxin H_(CN) domain played anintegral role in the cell binding step of the intoxication process.

Thus, the present invention discloses modified Clostridial toxins thatexhibit 1) an enhanced translocation capability; and 2) an enhancedtargeting capability for a naturally-occurring Clostridial toxinreceptor. The enhanced translocation capability is mediated by atranslocation facilitating domain comprising, e.g., a H_(CN) region ofClostridial toxins. The H_(CN) domain enhances the process by which theH_(N) domain mediates the release of the light chain from internalizedintracellular vesicles into the cytoplasm of the target cell during thetranslocation step. Enhanced translocation capability is obtained byincluding or maintaining a Clostridial toxin H_(CN) domain in a modifiedClostridial toxin disclosed in the present specification. The enhancedtargeting capability for a naturally-occurring Clostridial toxinreceptor is mediated by an enhanced targeting domain comprising amodified H_(CC) targeting domain of Clostridial toxins. The H_(CC)domain primarily determines the binding activity and binding specificityof the toxin to the receptor complex located at the surface of thetarget cell. Enhanced binding activity is achieved by replacing theendogenous H_(CC) targeting domain of a Clostridial toxin with atargeting domain showing enhanced binding activity for a Clostridialtoxin receptor present on a naturally-occurring Clostridial toxin targetcell.

Both the enhanced translocation and enhanced targeting activity shouldallow lower effective doses of a modified Clostridial toxin to beadministered to an individual because more toxin will be delivered to atarget cell. Thus modified Clostridial toxins with enhancedtranslocation and binding capabilities will reduce the undesirabledispersal of the toxin to areas not targeted for treatment, therebyreducing or preventing the undesirable side-effects associated withdiffusion of a Clostridial toxin to an unwanted location.

Thus, aspects of the present invention provide modified Clostridialtoxins comprising a Clostridial toxin enzymatic domain, a Clostridialtoxin translocation domain, a translocation facilitating domain and anenhanced targeting domain, wherein the modified Clostridial toxinexhibits an enhanced targeting activity for a Clostridial toxin receptoras compared to a naturally-occurring Clostridial toxin. It is envisionedthat any translocation facilitating domain capable of furtherfacilitating the translocation step of the intoxication process wherethe light chain is released from intracellular vesicles into thecytoplasm of the target cell will be useful to practice aspects of thepresent invention, including, without limitation, a Clostridial toxintranslocation facilitating domain and an enveloped virus fusogenicpeptide domain. Likewise, a multitude of enhanced targeting domains areenvisioned, including, without limitation, a modified Clostridial toxintargeting domain with enhanced binding activity, such as, e.g., a BoNT/Atargeting domain with enhanced binding activity, a BoNT/B targetingdomain with enhanced binding activity, a BoNT/C1 targeting domain withenhanced binding activity, a BoNT/D targeting domain with enhancedbinding activity, a BoNT/E targeting domain with enhanced bindingactivity, a BoNT/F targeting domain with enhanced binding activity, aBoNT/G targeting domain with enhanced binding activity, a TeNT targetingdomain with enhanced binding activity, or active fragment thereof; aClostridial NAP, such as, e.g., a Clostridial botulinum serotype AHA-33, a Clostridial botulinum serotype B HA-33, a Clostridial botulinumserotype C1 HA-33, a Clostridial botulinum serotype D HA-33, aClostridial botulinum serotype A HA-17, a Clostridial botulinum serotypeB HA-17, a Clostridial botulinum serotype C1 HA-17, a Clostridialbotulinum serotype D HA-17, a Clostridial botulinum serotype A NTNH, aClostridial botulinum serotype B NTNH, a Clostridial botulinum serotypeC1 NTNH, a Clostridial botulinum serotype D NTNH, a Clostridialbotulinum serotype E NTNH, a Clostridial botulinum serotype F NTNH and aClostridial botulinum serotype G NTNH; and a FGF, such as, e.g., aFGF-1, a FGF-2, a FGF-4, a FGF-8, a FGF-9, a FGF-17 and a FGF-18. It isalso envisioned that the location of the enhanced targeting domain inthe modified Clostridial toxins of the present specification can belocated at the amino terminus of the toxin, between the enzymatic andtranslocation domains or at the carboxyl terminus of the toxin. Thus, amodified Clostridial toxins disclosed in the present specification cancomprise an amino to carboxyl domain arrangement of, e.g., an enhancedtargeting domain, a Clostridial toxin translocation domain, atranslocation facilitating domain and a Clostridial toxin enzymaticdomain; an enhanced targeting domain, a Clostridial toxin enzymaticdomain, a Clostridial toxin translocation domain and a translocationfacilitating domain; a Clostridial toxin enzymatic domain, an enhancedtargeting domain, a Clostridial toxin translocation domain and atranslocation facilitating domain; a Clostridial toxin translocationdomain, a translocation facilitating domain, an enhanced targetingdomain and a Clostridial toxin enzymatic domain; a Clostridial toxinenzymatic domain, a Clostridial toxin translocation domain, atranslocation facilitating domain and an enhanced targeting domain; anda Clostridial toxin translocation domain, a translocation facilitatingdomain, a Clostridial toxin enzymatic domain and an enhanced targetingdomain.

Other aspects of the present invention provide polynucleotide moleculesencoding modified Clostridial toxins comprising a Clostridial toxinenzymatic domain, a Clostridial toxin translocation domain, atranslocation facilitating domain and an enhanced targeting domain,wherein the modified Clostridial toxin exhibits an enhanced targetingactivity for a Clostridial toxin receptor as compared to anaturally-occurring Clostridial toxin. It is envisioned that thelocation of the enhanced targeting domain of the modified Clostridialtoxins encoded by polynucleotide molecules of the present specificationcan be located at the amino terminus of the toxin, between the enzymaticand translocation domains or at the carboxyl terminus of the toxin.

Other aspects of the present invention provide methods of producing amodified Clostridial toxin disclosed in the present specification, themethod comprising the step of expressing in a cell a polynucleotidemolecule encoding a modified Clostridial toxin comprising a Clostridialtoxin enzymatic domain, a Clostridial toxin translocation domain, atranslocation facilitating domain and an enhanced targeting domain,wherein the modified Clostridial toxin exhibits an enhanced targetingactivity for a Clostridial toxin receptor as compared to anaturally-occurring Clostridial toxin. Other aspects of the presentinvention provide methods of producing a modified Clostridial toxindisclosed in the present specification, the method comprising the stepsof introducing in a cell an expression construct comprising apolynucleotide molecule encoding a modified Clostridial toxin comprisinga Clostridial toxin enzymatic domain, a Clostridial toxin translocationdomain, a translocation facilitating domain and an enhanced targetingdomain, wherein the modified Clostridial toxin exhibits an enhancedtargeting activity for a Clostridial toxin receptor as compared to anaturally-occurring Clostridial toxin and expressing the expressionconstruct in the cell.

Aspects of the present invention provide, in part, a modifiedClostridial toxin. As used herein, the term “modified Clostridial toxin”means any polypeptide that can execute the overall cellular mechanismwhereby a Clostridial toxin enters a neuron and inhibitsneurotransmitter release and encompasses the binding of a Clostridialtoxin to a low or high affinity receptor complex, the internalization ofthe toxin, the translocation of the Clostridial toxin light chain intothe cytoplasm and the enzymatic modification of a Clostridial toxinsubstrate. A modified Clostridial toxin disclosed in the presentspecification is distinguished from a naturally-occurring Clostridialtoxin by the fact that a modified Clostridial toxin comprises atranslocation facilitating domain that enhances the process by which alight chain from a modified toxin translocates into the cytoplasm of atarget cell and a modified Clostridial toxin lacks the cell bindingactivity of a naturally-occurring binding domain found in a Clostridialtoxin. Instead, a modified Clostridial toxin disclosed in the presentspecification comprises an enhanced targeting domain that determines thebinding activity of the modified Clostridial toxin to an endogenousClostridial toxin receptor located at the surface of the target cell. Bydefinition, a naturally-occurring Clostridial toxin lacks an enhancedtargeting domain. Examples of modified Clostridial toxin are describedin, e.g., Lance E. Steward, et al., Modified Clostridial Toxins withEnhanced Targeting Capabilities For Endogenous Clostridial ToxinReceptor Systems, International Patent Application No. 2006/008956 (Mar.14, 2006). Any of the modified Clostridial toxins described in, e.g.,Steward, supra, (Mar. 14, 2006), can be further modified to include atranslocation facilitating domain as disclosed in the presentspecification.

Aspects of the present invention provide, in part, a Clostridial toxinenzymatic domain. As used herein, the term “Clostridial toxin enzymaticdomain” means any Clostridial toxin polypeptide that can execute theenzymatic target modification step of the intoxication process. Thus, aClostridial toxin enzymatic domain specifically targets a Clostridialtoxin substrate and encompasses the proteolytic cleavage of aClostridial toxin substrate, such as, e.g., SNARE proteins like aSNAP-25 substrate, a VAMP substrate and a Syntaxin substrate.Non-limiting examples of a Clostridial toxin enzymatic domain include,e.g., a Clostridial toxin light chain region such as, e.g., a BoNT/Alight chain region, a BoNT/B light chain region, a BoNT/C1 light chainregion, a BoNT/D light chain region, a BoNT/E light chain region, aBoNT/F light chain region, a BoNT/G light chain region, and a TeNT lightchain region.

A Clostridial toxin enzymatic domain includes, without limitation,naturally occurring Clostridial toxin light chain variants, such as,e.g., Clostridial toxin light chain isoforms and Clostridial toxin lightchain subtypes; non-naturally occurring Clostridial toxin light chainvariants, such as, e.g., conservative Clostridial toxin light chainvariants, non-conservative Clostridial toxin light chain variants,Clostridial toxin light chain chimerics, active Clostridial toxin lightchain fragments thereof, or any combination thereof.

As used herein, the term “Clostridial toxin light chain variant,”whether naturally-occurring or non-naturally-occurring, means aClostridial toxin light chain that has at least one amino acid changefrom the corresponding region of the disclosed reference sequences (seeTable 1) and can be described in percent identity to the correspondingregion of that reference sequence. Unless expressly indicated, allClostridial toxin light chain variants disclosed in the presentspecification are capable of executing the enzymatic target modificationstep of the intoxication process. As non-limiting examples, a BoNT/Alight chain variant comprising amino acids 1-448 of SEQ ID NO: 1 willhave at least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region1-448 of SEQ ID NO: 1; a BoNT/B light chain variant comprising aminoacids 1-441 of SEQ ID NO: 2 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1-441 of SEQ ID NO: 2; aBoNT/C1 light chain variant comprising amino acids 1-449 of SEQ ID NO: 3will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-449 of SEQ ID NO: 3; a BoNT/D light chain variant comprisingamino acids 1-445 of SEQ ID NO: 4 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1-445 of SEQ ID NO: 4; aBoNT/E light chain variant comprising amino acids 1-422 of SEQ ID NO: 5will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-422 of SEQ ID NO: 5; a BoNT/F light chain variant comprisingamino acids 1-439 of SEQ ID NO: 6 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1-439 of SEQ ID NO: 6; aBoNT/G light chain variant comprising amino acids 1-446 of SEQ ID NO: 7will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-446 of SEQ ID NO: 7; and a TeNT light chain variant comprisingamino acids 1-457 of SEQ ID NO: 8 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1-457 of SEQ ID NO: 8.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxinlight chain variants that differ somewhat in their amino acid sequence,and also in the nucleic acids encoding these proteins. For example,there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 andBoNT/A4, with specific light chain subtypes showing approximately 95%amino acid identity when compared to another BoNT/A light chain subtype.As used herein, the term “naturally occurring Clostridial toxin lightchain variant” means any Clostridial toxin light chain produced by anaturally-occurring process, including, without limitation, Clostridialtoxin light chain isoforms produced from alternatively-splicedtranscripts, Clostridial toxin light chain isoforms produced byspontaneous mutation and Clostridial toxin light chain subtypes. Anaturally occurring Clostridial toxin light chain variant can functionin substantially the same manner as the reference Clostridial toxinlight chain on which the naturally occurring Clostridial toxin lightchain variant is based, and can be substituted for the referenceClostridial toxin light chain in any aspect of the present invention. Anaturally occurring Clostridial toxin light chain variant may substituteone or more amino acids, two or more amino acids, three or more aminoacids, four or more amino acids, five or more amino acids, ten or moreamino acids, 20 or more amino acids, 30 or more amino acids, 40 or moreamino acids, 50 or more amino acids or 100 or more amino acids from thereference Clostridial toxin light chain on which the naturally occurringClostridial toxin light chain variant is based. A naturally occurringClostridial toxin light chain variant can also substitute at least 10contiguous amino acids, at least 15 contiguous amino acids, at least 20contiguous amino acids, or at least 25 contiguous amino acids from thereference Clostridial toxin light chain on which the naturally occurringClostridial toxin light chain variant is based, that possess at least50% amino acid identity, 65% amino acid identity, 75% amino acididentity, 85% amino acid identity or 95% amino acid identity to thereference Clostridial toxin light chain on which the naturally occurringClostridial toxin light chain variant is based.

A non-limiting examples of a naturally occurring Clostridial toxin lightchain variant is a Clostridial toxin light chain isoform such as, e.g.,a BoNT/A light chain isoform, a BoNT/B light chain isoform, a BoNT/C1light chain isoform, a BoNT/D light chain isoform, a BoNT/E light chainisoform, a BoNT/F light chain isoform, a BoNT/G light chain isoform, anda TeNT light chain isoform. A Clostridial toxin light chain isoform canfunction in substantially the same manner as the reference Clostridialtoxin light chain on which the Clostridial toxin light chain isoform isbased, and can be substituted for the reference Clostridial toxin lightchain in any aspect of the present invention.

Another non-limiting examples of a naturally occurring Clostridial toxinlight chain variant is a Clostridial toxin light chain subtype such as,e.g., a light chain from subtype BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4;a light chain from subtype BoNT/B1, BoNT/B2, BoNT/B bivalent and BoNT/Bnonproteolytic; a light chain from subtype BoNT/C1-1 and BoNT/C1-2; alight chain from subtype BoNT/E1, BoNT/E2 and BoNT/E3; and a light chainfrom subtype BoNT/F1, BoNT/F2, BoNT/F3 and BoNT/F4. A Clostridial toxinlight chain subtype can function in substantially the same manner as thereference Clostridial toxin light chain on which the Clostridial toxinlight chain subtype is based, and can be substituted for the referenceClostridial toxin light chain in any aspect of the present invention.

As used herein, the term “non-naturally occurring Clostridial toxinlight chain variant” means any Clostridial toxin light chain producedwith the aid of human manipulation, including, without limitation,Clostridial toxin light chains produced by genetic engineering usingrandom mutagenesis or rational design and Clostridial toxin light chainsproduced by chemical synthesis. Non-limiting examples of non-naturallyoccurring Clostridial toxin light chain variants include, e.g.,conservative Clostridial toxin light chain variants, non-conservativeClostridial toxin light chain variants, Clostridial toxin light chainchimeric variants and active Clostridial toxin light chain fragments.

As used herein, the term “conservative Clostridial toxin light chainvariant” means a Clostridial toxin light chain that has at least oneamino acid substituted by another amino acid or an amino acid analogthat has at least one property similar to that of the original aminoacid from the reference Clostridial toxin light chain sequence (Table1). Examples of properties include, without limitation, similar size,topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservativeClostridial toxin light chain variant can function in substantially thesame manner as the reference Clostridial toxin light chain on which theconservative Clostridial toxin light chain variant is based, and can besubstituted for the reference Clostridial toxin light chain in anyaspect of the present invention. A conservative Clostridial toxin lightchain variant may substitute one or more amino acids, two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, ten or more amino acids, 20 or more amino acids, 30 or moreamino acids, 40 or more amino acids, 50 or more amino acids, 100 or moreamino acids or 200 or more amino acids from the reference Clostridialtoxin light chain on which the conservative Clostridial toxin lightchain variant is based. A conservative Clostridial toxin light chainvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference Clostridial toxinlight chain on which the conservative Clostridial toxin light chainvariant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial toxin light chainon which the conservative Clostridial toxin light chain variant isbased. Non-limiting examples of a conservative Clostridial toxin lightchain variant include, e.g., conservative BoNT/A light chain variants,conservative BoNT/B light chain variants, conservative BoNT/C1 lightchain variants, conservative BoNT/D light chain variants, conservativeBoNT/E light chain variants, conservative BoNT/F light chain variants,conservative BoNT/G light chain variants, and conservative TeNT lightchain variants.

As used herein, the term “non-conservative Clostridial toxin light chainvariant” means a Clostridial toxin light chain in which 1) at least oneamino acid is deleted from the reference Clostridial toxin light chainon which the non-conservative Clostridial toxin light chain variant isbased; 2) at least one amino acid added to the reference Clostridialtoxin light chain on which the non-conservative Clostridial toxin lightchain is based; or 3) at least one amino acid is substituted by anotheramino acid or an amino acid analog that does not share any propertysimilar to that of the original amino acid from the referenceClostridial toxin light chain sequence (Table 1). A non-conservativeClostridial toxin light chain variant can function in substantially thesame manner as the reference Clostridial toxin light chain on which thenon-conservative Clostridial toxin light chain variant is based, and canbe substituted for the reference Clostridial toxin light chain in anyaspect of the present invention. A non-conservative Clostridial toxinlight chain variant can delete one or more amino acids, two or moreamino acids, three or more amino acids, four or more amino acids, fiveor more amino acids, and ten or more amino acids from the referenceClostridial toxin light chain on which the non-conservative Clostridialtoxin light chain variant is based. A non-conservative Clostridial toxinlight chain variant can add one or more amino acids, two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, and ten or more amino acids to the reference Clostridialtoxin light chain on which the non-conservative Clostridial toxin lightchain variant is based. A non-conservative Clostridial toxin light chainvariant may substitute one or more amino acids, two or more amino acids,three or more amino acids, four or more amino acids, five or more aminoacids, ten or more amino acids, 20 or more amino acids, 30 or more aminoacids, 40 or more amino acids, 50 or more amino acids, 100 or more aminoacids or 200 or more amino acids from the reference Clostridial toxinlight chain on which the non-conservative Clostridial toxin light chainvariant is based. A non-conservative Clostridial toxin light chainvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference Clostridial toxinlight chain on which the non-conservative Clostridial toxin light chainvariant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial toxin light chainon which the non-conservative Clostridial toxin light chain variant isbased. Non-limiting examples of a non-conservative Clostridial toxinlight chain variant include, e.g., non-conservative BoNT/A light chainvariants, non-conservative BoNT/B light chain variants, non-conservativeBoNT/C1 light chain variants, non-conservative BoNT/D light chainvariants, non-conservative BoNT/E light chain variants, non-conservativeBoNT/F light chain variants, non-conservative BoNT/G light chainvariants, and non-conservative TeNT light chain variants.

As used herein, the term “Clostridial toxin light chain chimeric” meansa polypeptide comprising at least a portion of a Clostridial toxin lightchain and at least a portion of at least one other polypeptide to form atoxin light chain with at least one property different from thereference Clostridial toxin light chains of Table 1, with the provisothat this Clostridial toxin light chain chimeric is still capable ofspecifically targeting the core components of the neurotransmitterrelease apparatus and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. Such Clostridial toxin light chain chimerics are describedin, e.g., Lance E. Steward et al., Leucine-based Motif and ClostridialToxins, U.S. Patent Publication 2003/0027752 (Feb. 6, 2003); Lance E.Steward et al., Clostridial Neurotoxin Compositions and ModifiedClostridial Neurotoxins, U.S. Patent Publication 2003/0219462 (Nov. 27,2003); and Lance E. Steward et al., Clostridial Neurotoxin Compositionsand Modified Clostridial Neurotoxins, U.S. Patent Publication2004/0220386 (Nov. 4, 2004).

As used herein, the term “active Clostridial toxin light chain fragment”means any of a variety of Clostridial toxin fragments comprising thelight chain can be useful in aspects of the present invention with theproviso that these light chain fragments can specifically target thecore components of the neurotransmitter release apparatus and thusparticipate in executing the overall cellular mechanism whereby aClostridial toxin proteolytically cleaves a substrate. The light chainsof Clostridial toxins are approximately 420-460 amino acids in lengthand comprise an enzymatic domain (Table 1). Research has shown that theentire length of a Clostridial toxin light chain is not necessary forthe enzymatic activity of the enzymatic domain. As a non-limitingexample, the first eight amino acids of the BoNT/A light chain (residues1-8 of SEQ ID NO: 1) are not required for enzymatic activity. As anothernon-limiting example, the first eight amino acids of the TeNT lightchain (residues 1-8 of SEQ ID NO: 8) are not required for enzymaticactivity. Likewise, the carboxyl-terminus of the light chain is notnecessary for activity. As a non-limiting example, the last 32 aminoacids of the BoNT/A light chain (residues 417-448 of SEQ ID NO: 1) arenot required for enzymatic activity. As another non-limiting example,the last 31 amino acids of the TeNT light chain (residues 427-457 of SEQID NO: 8) are not required for enzymatic activity. Thus, aspects of thisembodiment can include Clostridial toxin light chains comprising anenzymatic domain having a length of, e.g., at least 350 amino acids, atleast 375 amino acids, at least 400 amino acids, at least 425 aminoacids and at least 450 amino acids. Other aspects of this embodiment caninclude Clostridial toxin light chains comprising an enzymatic domainhaving a length of, e.g., at most 350 amino acids, at most 375 aminoacids, at most 400 amino acids, at most 425 amino acids and at most 450amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin light chainvariants and non-naturally-occurring Clostridial toxin light chainvariants, including, without limitation, global methods, local methodsand hybrid methods, such as, e.g., segment approach methods. Protocolsto determine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

Global methods align sequences from the beginning to the end of themolecule and determine the best alignment by adding up scores ofindividual residue pairs and by imposing gap penalties. Non-limitingmethods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al.,CLUSTAL W: Improving the Sensitivity of Progressive Multiple SequenceAlignment Through Sequence Weighting, Position-Specific Gap Penaltiesand Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680(1994); and iterative refinement, see, e.g., Osamu Gotoh, SignificantImprovement in Accuracy of Multiple Protein Sequence Alignments byIterative Refinement as Assessed by Reference to Structural Alignments,264(4) J. Mol. Biol. 823-838 (1996).

Local methods align sequences by identifying one or more conservedmotifs shared by all of the input sequences. Non-limiting methodsinclude, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans,Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignmentof Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbssampling, see, e.g., C. E. Lawrence et al., Detecting Subtle SequenceSignals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131)Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al.,Align-M—A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20(9) Bioinformatics:1428-1435 (2004).

Hybrid methods combine functional aspects of both global and localalignment methods. Non-limiting methods include, e.g.,segment-to-segment comparison, see, e.g., Burkhard Morgenstern et al.,Multiple DNA and Protein Sequence Alignment Based On Segment-To-SegmentComparison, 93(22) Proc. Natl. Acad. Sci. U.S.A. 12098-12103 (1996);T-Coffee, see, e.g., Cédric Notredame et al., T-Coffee: A NovelAlgorithm for Multiple Sequence Alignment, 302(1) J. Mol. Biol. 205-217(2000); MUSCLE, see, e.g., Robert C. Edgar, MUSCLE: Multiple SequenceAlignment With High Score Accuracy and High Throughput, 32(5) NucleicAcids Res. 1792-1797 (2004); and DIALIGN-T, see, e.g., Amarendran RSubramanian et al., DIALIGN-T: An Improved Algorithm for Segment-BasedMultiple Sequence Alignment, 6(1) BMC Bioinformatics 66 (2005).

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin enzymatic domain. Inan aspect of this embodiment, a Clostridial toxin enzymatic domaincomprises a naturally occurring Clostridial toxin light chain variant,such as, e.g., a Clostridial toxin light chain isoform or a Clostridialtoxin light chain subtype. In another aspect of this embodiment, aClostridial toxin enzymatic domain comprises a non-naturally occurringClostridial toxin light chain variant, such as, e.g., a conservativeClostridial toxin light chain variant, a non-conservative Clostridialtoxin light chain variant, a Clostridial toxin chimeric light chain, anactive Clostridial toxin light chain fragment, or any combinationthereof.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/A light chain. In an aspect of this embodiment, a BoNT/A lightchain comprises amino acids 1-448 of SEQ ID NO: 1. In another aspect ofthis embodiment, a BoNT/A light chain comprises a naturally occurringBoNT/A light chain variant, such as, e.g., a light chain from a BoNT/Aisoform or a light chain from a BoNT/A subtype. In another aspect ofthis embodiment, a BoNT/A light chain comprises amino acids 1-448 of anaturally occurring BoNT/A light chain variant of SEQ ID NO: 1, such as,e.g., amino acids 1-448 of a BoNT/A isoform of SEQ ID NO: 1 or aminoacids 1-448 of a BoNT/A subtype of SEQ ID NO: 1. In still another aspectof this embodiment, a BoNT/A light chain comprises a non-naturallyoccurring BoNT/A light chain variant, such as, e.g., a conservativeBoNT/A light chain variant, a non-conservative BoNT/A light chainvariant, a BoNT/A chimeric light chain, an active BoNT/A light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/A light chain comprises amino acids 1-448 of anon-naturally occurring BoNT/A light chain variant of SEQ ID NO: 1, suchas, e.g., amino acids 1-448 of a conservative BoNT/A light chain variantof SEQ ID NO: 1, amino acids 1-448 of a non-conservative BoNT/A lightchain variant of SEQ ID NO: 1, amino acids 1-448 of an active BoNT/Alight chain fragment of SEQ ID NO: 1, or any combination thereof.

In other aspects of this embodiment, a BoNT/A light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 75% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 80% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 85% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1, at least 90% amino acid identity with aminoacids 1-448 of SEQ ID NO: 1 or at least 95% amino acid identity withamino acids 1-448 of SEQ ID NO: 1. In yet other aspects of thisembodiment, a BoNT/A light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-448 of SEQ ID NO: 1,at most 75% amino acid identity with amino acids 1-448 of SEQ ID NO: 1,at most 80% amino acid identity with amino acids 1-448 of SEQ ID NO: 1,at most 85% amino acid identity with amino acids 1-448 of SEQ ID NO: 1,at most 90% amino acid identity with amino acids 1-448 of SEQ ID NO: 1or at most 95% amino acid identity with amino acids 1-448 of SEQ ID NO:1.

In other aspects of this embodiment, a BoNT/A light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-448 of SEQ ID NO: 1. Inother aspects of this embodiment, a BoNT/A light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-448 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-448 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-448 of SEQ ID NO: 1. In still other aspects ofthis embodiment, a BoNT/A light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-448 of SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-448 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-448 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-448 of SEQ ID NO: 1. In yet other aspects ofthis embodiment, a BoNT/A light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-448 of SEQ ID NO: 1. In other aspects of this embodiment,a BoNT/A light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-448 ofSEQ ID NO: 1. In still other aspects of this embodiment, a BoNT/A lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-448 of SEQ IDNO: 1. In other aspects of this embodiment, a BoNT/A light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-448 of SEQ ID NO: 1.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/B light chain. In an aspect of this embodiment, a BoNT/B lightchain comprises amino acids 1-441 of SEQ ID NO: 2. In another aspect ofthis embodiment, a BoNT/B light chain comprises a naturally occurringBoNT/B light chain variant, such as, e.g., a light chain from a BoNT/Bisoform or a light chain from a BoNT/B subtype. In another aspect ofthis embodiment, a BoNT/B light chain comprises amino acids 1-441 of anaturally occurring BoNT/B light chain variant of SEQ ID NO: 2, such as,e.g., amino acids 1-441 of a BoNT/B isoform of SEQ ID NO: 2 or aminoacids 1-441 of a BoNT/B subtype of SEQ ID NO: 2. In still another aspectof this embodiment, a BoNT/B light chain comprises a non-naturallyoccurring BoNT/B light chain variant, such as, e.g., a conservativeBoNT/B light chain variant, a non-conservative BoNT/B light chainvariant, a BoNT/B chimeric light chain, an active BoNT/B light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/B light chain comprises amino acids 1-441 of anon-naturally occurring BoNT/B light chain variant of SEQ ID NO: 2, suchas, e.g., amino acids 1-441 of a conservative BoNT/B light chain variantof SEQ ID NO: 2, amino acids 1-441 of a non-conservative BoNT/B lightchain variant of SEQ ID NO: 2, amino acids 1-441 of an active BoNT/Blight chain fragment of SEQ ID NO: 2, or any combination thereof.

In other aspects of this embodiment, a BoNT/B light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 75% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 80% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 85% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2, at least 90% amino acid identity with aminoacids 1-441 of SEQ ID NO: 2 or at least 95% amino acid identity withamino acids 1-441 of SEQ ID NO: 2. In yet other aspects of thisembodiment, a BoNT/B light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-441 of SEQ ID NO: 2,at most 75% amino acid identity with amino acids 1-441 of SEQ ID NO: 2,at most 80% amino acid identity with amino acids 1-441 of SEQ ID NO: 2,at most 85% amino acid identity with amino acids 1-441 of SEQ ID NO: 2,at most 90% amino acid identity with amino acids 1-441 of SEQ ID NO: 2or at most 95% amino acid identity with amino acids 1-441 of SEQ ID NO:2.

In other aspects of this embodiment, a BoNT/B light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-441 of SEQ ID NO: 2. Inother aspects of this embodiment, a BoNT/B light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-441 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-441 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-441 of SEQ ID NO: 2. In still other aspects ofthis embodiment, a BoNT/B light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-441 of SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-441 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-441 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-441 of SEQ ID NO: 2. In yet other aspects ofthis embodiment, a BoNT/B light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-441 of SEQ ID NO: 2. In other aspects of this embodiment,a BoNT/B light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-441 ofSEQ ID NO: 2. In still other aspects of this embodiment, a BoNT/B lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-441 of SEQ IDNO: 2. In other aspects of this embodiment, a BoNT/B light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-441 of SEQ ID NO: 2.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/C1 light chain. In an aspect of this embodiment, a BoNT/C1 lightchain comprises amino acids 1-449 of SEQ ID NO: 3. In another aspect ofthis embodiment, a BoNT/C1 light chain comprises a naturally occurringBoNT/C1 light chain variant, such as, e.g., a light chain from a BoNT/C1isoform or a light chain from a BoNT/C1 subtype. In another aspect ofthis embodiment, a BoNT/C1 light chain comprises amino acids 1-449 of anaturally occurring BoNT/C1 light chain variant of SEQ ID NO: 3, suchas, e.g., amino acids 1-449 of a BoNT/C1 isoform of SEQ ID NO: 3 oramino acids 1-449 of a BoNT/C1 subtype of SEQ ID NO: 3. In still anotheraspect of this embodiment, a BoNT/C1 light chain comprises anon-naturally occurring BoNT/C1 light chain variant, such as, e.g., aconservative BoNT/C1 light chain variant, a non-conservative BoNT/C1light chain variant, a BoNT/C1 chimeric light chain, an active BoNT/C1light chain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/C1 light chain comprises amino acids1-449 of a non-naturally occurring BoNT/C1 light chain variant of SEQ IDNO: 3, such as, e.g., amino acids 1-449 of a conservative BoNT/C1 lightchain variant of SEQ ID NO: 3, amino acids 1-449 of a non-conservativeBoNT/C1 light chain variant of SEQ ID NO: 3, amino acids 1-449 of anactive BoNT/C1 light chain fragment of SEQ ID NO: 3, or any combinationthereof.

In other aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-449 of SEQ ID NO: 3, at least 75% amino acid identity with aminoacids 1-449 of SEQ ID NO: 3, at least 80% amino acid identity with aminoacids 1-449 of SEQ ID NO: 3, at least 85% amino acid identity with aminoacids 1-449 of SEQ ID NO: 3, at least 90% amino acid identity with aminoacids 1-449 of SEQ ID NO: 3 or at least 95% amino acid identity withamino acids 1-449 of SEQ ID NO: 3. In yet other aspects of thisembodiment, a BoNT/C1 light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-449 of SEQ ID NO: 3,at most 75% amino acid identity with amino acids 1-449 of SEQ ID NO: 3,at most 80% amino acid identity with amino acids 1-449 of SEQ ID NO: 3,at most 85% amino acid identity with amino acids 1-449 of SEQ ID NO: 3,at most 90% amino acid identity with amino acids 1-449 of SEQ ID NO: 3or at most 95% amino acid identity with amino acids 1-449 of SEQ ID NO:3.

In other aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-449 of SEQ ID NO: 3. Inother aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-449 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-449 of SEQ ID NO: 3. In stillother aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 1-449 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-449 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 1-449 of SEQ ID NO: 3. In still otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-449 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-449 of SEQ ID NO: 3.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/D light chain. In an aspect of this embodiment, a BoNT/D lightchain comprises amino acids 1-445 of SEQ ID NO: 4. In another aspect ofthis embodiment, a BoNT/D light chain comprises a naturally occurringBoNT/D light chain variant, such as, e.g., a light chain from a BoNT/Disoform or a light chain from a BoNT/D subtype. In another aspect ofthis embodiment, a BoNT/D light chain comprises amino acids 1-445 of anaturally occurring BoNT/D light chain variant of SEQ ID NO: 4, such as,e.g., amino acids 1-445 of a BoNT/D isoform of SEQ ID NO: 4 or aminoacids 1-445 of a BoNT/D subtype of SEQ ID NO: 4. In still another aspectof this embodiment, a BoNT/D light chain comprises a non-naturallyoccurring BoNT/D light chain variant, such as, e.g., a conservativeBoNT/D light chain variant, a non-conservative BoNT/D light chainvariant, a BoNT/D chimeric light chain, an active BoNT/D light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/D light chain comprises amino acids 1-445 of anon-naturally occurring BoNT/D light chain variant of SEQ ID NO: 4, suchas, e.g., amino acids 1-445 of a conservative BoNT/D light chain variantof SEQ ID NO: 4, amino acids 1-445 of a non-conservative BoNT/D lightchain variant of SEQ ID NO: 4, amino acids 1-445 of an active BoNT/Dlight chain fragment of SEQ ID NO: 4, or any combination thereof.

In other aspects of this embodiment, a BoNT/D light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 75% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 80% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 85% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4, at least 90% amino acid identity with aminoacids 1-445 of SEQ ID NO: 4 or at least 95% amino acid identity withamino acids 1-445 of SEQ ID NO: 4. In yet other aspects of thisembodiment, a BoNT/D light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-445 of SEQ ID NO: 4,at most 75% amino acid identity with amino acids 1-445 of SEQ ID NO: 4,at most 80% amino acid identity with amino acids 1-445 of SEQ ID NO: 4,at most 85% amino acid identity with amino acids 1-445 of SEQ ID NO: 4,at most 90% amino acid identity with amino acids 1-445 of SEQ ID NO: 4or at most 95% amino acid identity with amino acids 1-445 of SEQ ID NO:4.

In other aspects of this embodiment, a BoNT/D light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-445 of SEQ ID NO: 4. Inother aspects of this embodiment, a BoNT/D light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-445 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-445 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-445 of SEQ ID NO: 4. In still other aspects ofthis embodiment, a BoNT/D light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-445 of SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-445 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-445 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-445 of SEQ ID NO: 4. In yet other aspects ofthis embodiment, a BoNT/D light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-445 of SEQ ID NO: 4. In other aspects of this embodiment,a BoNT/D light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-445 ofSEQ ID NO: 4. In still other aspects of this embodiment, a BoNT/D lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-445 of SEQ IDNO: 4. In other aspects of this embodiment, a BoNT/D light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-445 of SEQ ID NO: 4.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/E light chain. In an aspect of this embodiment, a BoNT/E lightchain comprises amino acids 1-422 of SEQ ID NO: 5. In another aspect ofthis embodiment, a BoNT/E light chain comprises a naturally occurringBoNT/E light chain variant, such as, e.g., a light chain from a BoNT/Eisoform or a light chain from a BoNT/E subtype. In another aspect ofthis embodiment, a BoNT/E light chain comprises amino acids 1-422 of anaturally occurring BoNT/E light chain variant of SEQ ID NO: 5, such as,e.g., amino acids 1-422 of a BoNT/E isoform of SEQ ID NO: 5 or aminoacids 1-422 of a BoNT/E subtype of SEQ ID NO: 5. In still another aspectof this embodiment, a BoNT/E light chain comprises a non-naturallyoccurring BoNT/E light chain variant, such as, e.g., a conservativeBoNT/E light chain variant, a non-conservative BoNT/E light chainvariant, a BoNT/E chimeric light chain, an active BoNT/E light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/E light chain comprises amino acids 1-422 of anon-naturally occurring BoNT/E light chain variant of SEQ ID NO: 5, suchas, e.g., amino acids 1-422 of a conservative BoNT/E light chain variantof SEQ ID NO: 5, amino acids 1-422 of a non-conservative BoNT/E lightchain variant of SEQ ID NO: 5, amino acids 1-422 of an active BoNT/Elight chain fragment of SEQ ID NO: 5, or any combination thereof.

In other aspects of this embodiment, a BoNT/E light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 75% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 80% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 85% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5, at least 90% amino acid identity with aminoacids 1-422 of SEQ ID NO: 5 or at least 95% amino acid identity withamino acids 1-422 of SEQ ID NO: 5. In yet other aspects of thisembodiment, a BoNT/E light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-422 of SEQ ID NO: 5,at most 75% amino acid identity with amino acids 1-422 of SEQ ID NO: 5,at most 80% amino acid identity with amino acids 1-422 of SEQ ID NO: 5,at most 85% amino acid identity with amino acids 1-422 of SEQ ID NO: 5,at most 90% amino acid identity with amino acids 1-422 of SEQ ID NO: 5or at most 95% amino acid identity with amino acids 1-422 of SEQ ID NO:5.

In other aspects of this embodiment, a BoNT/E light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-422 of SEQ ID NO: 5. Inother aspects of this embodiment, a BoNT/E light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-422 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-422 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-422 of SEQ ID NO: 5. In still other aspects ofthis embodiment, a BoNT/E light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-422 of SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-422 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-422 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-422 of SEQ ID NO: 5. In yet other aspects ofthis embodiment, a BoNT/E light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-422 of SEQ ID NO: 5. In other aspects of this embodiment,a BoNT/E light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-422 ofSEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/E lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-422 of SEQ IDNO: 5. In other aspects of this embodiment, a BoNT/E light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-422 of SEQ ID NO: 5.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/F light chain. In an aspect of this embodiment, a BoNT/F lightchain comprises amino acids 1-439 of SEQ ID NO: 6. In another aspect ofthis embodiment, a BoNT/F light chain comprises a naturally occurringBoNT/F light chain variant, such as, e.g., a light chain from a BoNT/Fisoform or a light chain from a BoNT/F subtype. In another aspect ofthis embodiment, a BoNT/F light chain comprises amino acids 1-439 of anaturally occurring BoNT/F light chain variant of SEQ ID NO: 6, such as,e.g., amino acids 1-439 of a BoNT/F isoform of SEQ ID NO: 6 or aminoacids 1-439 of a BoNT/F subtype of SEQ ID NO: 6. In still another aspectof this embodiment, a BoNT/F light chain comprises a non-naturallyoccurring BoNT/F light chain variant, such as, e.g., a conservativeBoNT/F light chain variant, a non-conservative BoNT/F light chainvariant, a BoNT/F chimeric light chain, an active BoNT/F light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/F light chain comprises amino acids 1-439 of anon-naturally occurring BoNT/F light chain variant of SEQ ID NO: 6, suchas, e.g., amino acids 1-439 of a conservative BoNT/F light chain variantof SEQ ID NO: 6, amino acids 1-439 of a non-conservative BoNT/F lightchain variant of SEQ ID NO: 6, amino acids 1-439 of an active BoNT/Flight chain fragment of SEQ ID NO: 6, or any combination thereof.

In other aspects of this embodiment, a BoNT/F light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 75% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 80% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 85% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6, at least 90% amino acid identity with aminoacids 1-439 of SEQ ID NO: 6 or at least 95% amino acid identity withamino acids 1-439 of SEQ ID NO: 6. In yet other aspects of thisembodiment, a BoNT/F light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-439 of SEQ ID NO: 6,at most 75% amino acid identity with amino acids 1-439 of SEQ ID NO: 6,at most 80% amino acid identity with amino acids 1-439 of SEQ ID NO: 6,at most 85% amino acid identity with amino acids 1-439 of SEQ ID NO: 6,at most 90% amino acid identity with amino acids 1-439 of SEQ ID NO: 6or at most 95% amino acid identity with amino acids 1-439 of SEQ ID NO:6.

In other aspects of this embodiment, a BoNT/F light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-439 of SEQ ID NO: 6. Inother aspects of this embodiment, a BoNT/F light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-439 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-439 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-439 of SEQ ID NO: 6. In still other aspects ofthis embodiment, a BoNT/F light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-439 of SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-439 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-439 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-439 of SEQ ID NO: 6. In yet other aspects ofthis embodiment, a BoNT/F light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-439 of SEQ ID NO: 6. In other aspects of this embodiment,a BoNT/F light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-439 ofSEQ ID NO: 6. In still other aspects of this embodiment, a BoNT/F lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-439 of SEQ IDNO: 6. In other aspects of this embodiment, a BoNT/F light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-439 of SEQ ID NO: 6.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/G light chain. In an aspect of this embodiment, a BoNT/G lightchain comprises amino acids 1-446 of SEQ ID NO: 7. In another aspect ofthis embodiment, a BoNT/G light chain comprises a naturally occurringBoNT/G light chain variant, such as, e.g., a light chain from a BoNT/Gisoform or a light chain from a BoNT/G subtype. In another aspect ofthis embodiment, a BoNT/G light chain comprises amino acids 1-446 of anaturally occurring BoNT/G light chain variant of SEQ ID NO: 7, such as,e.g., amino acids 1-446 of a BoNT/G isoform of SEQ ID NO: 7 or aminoacids 1-446 of a BoNT/G subtype of SEQ ID NO: 7. In still another aspectof this embodiment, a BoNT/G light chain comprises a non-naturallyoccurring BoNT/G light chain variant, such as, e.g., a conservativeBoNT/G light chain variant, a non-conservative BoNT/G light chainvariant, a BoNT/G chimeric light chain, an active BoNT/G light chainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/G light chain comprises amino acids 1-446 of anon-naturally occurring BoNT/G light chain variant of SEQ ID NO: 7, suchas, e.g., amino acids 1-446 of a conservative BoNT/G light chain variantof SEQ ID NO: 7, amino acids 1-446 of a non-conservative BoNT/G lightchain variant of SEQ ID NO: 7, amino acids 1-446 of an active BoNT/Glight chain fragment of SEQ ID NO: 7, or any combination thereof.

In other aspects of this embodiment, a BoNT/G light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 75% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 80% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 85% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7, at least 90% amino acid identity with aminoacids 1-446 of SEQ ID NO: 7 or at least 95% amino acid identity withamino acids 1-446 of SEQ ID NO: 7. In yet other aspects of thisembodiment, a BoNT/G light chain comprises a polypeptide having, e.g.,at most 70% amino acid identity with amino acids 1-446 of SEQ ID NO: 7,at most 75% amino acid identity with amino acids 1-446 of SEQ ID NO: 7,at most 80% amino acid identity with amino acids 1-446 of SEQ ID NO: 7,at most 85% amino acid identity with amino acids 1-446 of SEQ ID NO: 7,at most 90% amino acid identity with amino acids 1-446 of SEQ ID NO: 7or at most 95% amino acid identity with amino acids 1-446 of SEQ ID NO:7.

In other aspects of this embodiment, a BoNT/G light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-446 of SEQ ID NO: 7. Inother aspects of this embodiment, a BoNT/G light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-446 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-446 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-446 of SEQ ID NO: 7. In still other aspects ofthis embodiment, a BoNT/G light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-446 of SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G light chain comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50, 100 or 200 non-contiguous amino acid additions relative toamino acids 1-446 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-446 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-446 of SEQ ID NO: 7. In yet other aspects ofthis embodiment, a BoNT/G light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-446 of SEQ ID NO: 7. In other aspects of this embodiment,a BoNT/G light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-446 ofSEQ ID NO: 7. In still other aspects of this embodiment, a BoNT/G lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-446 of SEQ IDNO: 7. In other aspects of this embodiment, a BoNT/G light chaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguousamino acid additions relative to amino acids 1-446 of SEQ ID NO: 7.

In another embodiment, a Clostridial toxin enzymatic domain comprises aTeNT light chain. In an aspect of this embodiment, a TeNT light chaincomprises amino acids 1-457 of SEQ ID NO: 8. In another aspect of thisembodiment, a TeNT light chain comprises a naturally occurring TeNTlight chain variant, such as, e.g., a light chain from a TeNT isoform ora light chain from a TeNT subtype. In another aspect of this embodiment,a TeNT light chain comprises amino acids 1-457 of a naturally occurringTeNT light chain variant of SEQ ID NO: 8, such as, e.g., amino acids1-457 of a TeNT isoform of SEQ ID NO: 8 or amino acids 1-457 of a TeNTsubtype of SEQ ID NO: 8. In still another aspect of this embodiment, aTeNT light chain comprises a non-naturally occurring TeNT light chainvariant, such as, e.g., a conservative TeNT light chain variant, anon-conservative TeNT light chain variant, a TeNT chimeric light chain,an active TeNT light chain fragment, or any combination thereof. Instill another aspect of this embodiment, a TeNT light chain comprisesamino acids 1-457 of a non-naturally occurring TeNT light chain variantof SEQ ID NO: 8, such as, e.g., amino acids 1-457 of a conservative TeNTlight chain variant of SEQ ID NO: 8, amino acids 1-457 of anon-conservative TeNT light chain variant of SEQ ID NO: 8, amino acids1-457 of an active TeNT light chain fragment of SEQ ID NO: 8, or anycombination thereof.

In other aspects of this embodiment, a TeNT light chain comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 75% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 80% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 85% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8, at least 90% amino acid identity with aminoacids 1-457 of SEQ ID NO: 8 or at least 95% amino acid identity withamino acids 1-457 of SEQ ID NO: 8. In yet other aspects of thisembodiment, a TeNT light chain comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 1-457 of SEQ ID NO: 8, atmost 75% amino acid identity with amino acids 1-457 of SEQ ID NO: 8, atmost 80% amino acid identity with amino acids 1-457 of SEQ ID NO: 8, atmost 85% amino acid identity with amino acids 1-457 of SEQ ID NO: 8, atmost 90% amino acid identity with amino acids 1-457 of SEQ ID NO: 8 orat most 95% amino acid identity with amino acids 1-457 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 1-457 of SEQ ID NO: 8. Inother aspects of this embodiment, a TeNT light chain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 1-457 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 1-457 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 1-457 of SEQ ID NO: 8. In still other aspects ofthis embodiment, a TeNT light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 1-457 of SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT light chain comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid additions relative to aminoacids 1-457 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT light chain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 1-457 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT light chain comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 1-457 of SEQ ID NO: 8. In yet other aspects ofthis embodiment, a TeNT light chain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 1-457 of SEQ ID NO: 8. In other aspects of this embodiment,a TeNT light chain comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid deletions relative to amino acids 1-457 ofSEQ ID NO: 8. In still other aspects of this embodiment, a TeNT lightchain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200contiguous amino acid additions relative to amino acids 1-457 of SEQ IDNO: 8. In other aspects of this embodiment, a TeNT light chain comprisesa polypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 1-457 of SEQ ID NO: 8.

Aspects of the present invention provide, in part, a Clostridial toxintranslocation domain. As used herein, the term “Clostridial toxintranslocation domain” means any Clostridial toxin polypeptide that canexecute the translocation step of the intoxication process that mediatesClostridial toxin light chain translocation. Thus, a Clostridial toxintranslocation domain facilitates the movement of a Clostridial toxinlight chain across a membrane and encompasses the movement of aClostridial toxin light chain through the membrane an intracellularvesicle into the cytoplasm of a cell. Non-limiting examples of aClostridial toxin translocation domain include, e.g., a Clostridialtoxin H_(N) region such as, e.g., a BoNT/A H_(N) region, a BoNT/B H_(N)region, a BoNT/C1 H_(N) region, a BoNT/D H_(N) region, a BoNT/E H_(N)region, a BoNT/F H_(N) region, a BoNT/G H_(N) region, and a TeNT H_(N)region.

A Clostridial toxin translocation domain includes, without limitation,naturally occurring Clostridial toxin H_(N) region variants, such as,e.g., Clostridial toxin H_(N) region isoforms and Clostridial toxinH_(N) region subtypes; non-naturally occurring Clostridial toxin H_(N)region variants, such as, e.g., conservative Clostridial toxin H_(N)region variants, non-conservative Clostridial toxin H_(N) regionvariants, Clostridial toxin H_(N) region chimerics, active Clostridialtoxin H_(N) region fragments thereof, or any combination thereof.

As used herein, the term “Clostridial toxin H_(N) region variant,”whether naturally-occurring or non-naturally-occurring, means aClostridial toxin H_(N) region that has at least one amino acid changefrom the corresponding region of the disclosed reference sequences (seeTable 1) and can be described in percent identity to the correspondingregion of that reference sequence. Unless expressly indicated, allClostridial toxin H_(N) region variants disclosed in the presentspecification are capable of executing the translocation step of theintoxication process that mediates Clostridial toxin light chaintranslocation. As non-limiting examples, a BoNT/A H_(N) region variantcomprising amino acids 449-873 of SEQ ID NO: 1 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 449-873 ofSEQ ID NO: 1; a BoNT/B H_(N) region variant comprising amino acids442-860 of SEQ ID NO: 2 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 442-860 of SEQ ID NO: 2; a BoNT/C1H_(N) region variant comprising amino acids 450-868 of SEQ ID NO: 3 willhave at least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region450-868 of SEQ ID NO: 3; a BoNT/D H_(N) region variant comprising aminoacids 446-864 of SEQ ID NO: 4 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 446-864 of SEQ ID NO: 4;a BoNT/E H_(N) region variant comprising amino acids 423-847 of SEQ IDNO: 5 will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to the aminoacid region 423-847 of SEQ ID NO: 5; a BoNT/F H_(N) region variantcomprising amino acids 440-866 of SEQ ID NO: 6 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 440-866 ofSEQ ID NO: 6; a BoNT/G H_(N) region variant comprising amino acids447-865 of SEQ ID NO: 7 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 447-865 of SEQ ID NO: 7; and a TeNTH_(N) region variant comprising amino acids 458-881 of SEQ ID NO: 8 willhave at least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region458-881 of SEQ ID NO: 8.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxinH_(N) region variants that differ somewhat in their amino acid sequence,and also in the nucleic acids encoding these proteins. For example,there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 andBoNT/A4, with specific H_(N) region subtypes showing approximately 87%amino acid identity when compared to another BoNT/A H_(N) regionsubtype. As used herein, the term “naturally occurring Clostridial toxinH_(N) region variant” means any Clostridial toxin H_(N) region producedby a naturally-occurring process, including, without limitation,Clostridial toxin H_(N) region isoforms produced fromalternatively-spliced transcripts, Clostridial toxin H_(N) regionisoforms produced by spontaneous mutation and Clostridial toxin H_(N)region subtypes. A naturally occurring Clostridial toxin H_(N) regionvariant can function in substantially the same manner as the referenceClostridial toxin H_(N) region on which the naturally occurringClostridial toxin H_(N) region variant is based, and can be substitutedfor the reference Clostridial toxin H_(N) region in any aspect of thepresent invention. A naturally occurring Clostridial toxin H_(N) regionvariant may substitute one or more amino acids, two or more amino acids,three or more amino acids, four or more amino acids, five or more aminoacids, ten or more amino acids, 20 or more amino acids, 30 or more aminoacids, 40 or more amino acids, 50 or more amino acids or 100 or moreamino acids from the reference Clostridial toxin H_(N) region on whichthe naturally occurring Clostridial toxin H_(N) region variant is based.A naturally occurring Clostridial toxin H_(N) region variant can alsosubstitute at least 10 contiguous amino acids, at least 15 contiguousamino acids, at least 20 contiguous amino acids, or at least 25contiguous amino acids from the reference Clostridial toxin H_(N) regionon which the naturally occurring Clostridial toxin H_(N) region variantis based, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference Clostridial toxin H_(N) region on whichthe naturally occurring Clostridial toxin H_(N) region variant is based.

A non-limiting examples of a naturally occurring Clostridial toxin H_(N)region variant is a Clostridial toxin H_(N) region isoform such as,e.g., a BoNT/A H_(N) region isoform, a BoNT/B H_(N) region isoform, aBoNT/C1 H_(N) region isoform, a BoNT/D H_(N) region isoform, a BoNT/EH_(N) region isoform, a BoNT/F H_(N) region isoform, a BoNT/G H_(N)region isoform, and a TeNT H_(N) region isoform. A Clostridial toxinH_(N) region isoform can function in substantially the same manner asthe reference Clostridial toxin H_(N) region on which the Clostridialtoxin H_(N) region isoform is based, and can be substituted for thereference Clostridial toxin H_(N) region in any aspect of the presentinvention.

Another non-limiting examples of a naturally occurring Clostridial toxinH_(N) region variant is a Clostridial toxin H_(N) region subtype suchas, e.g., a H_(N) region from subtype BoNT/A1, BoNT/A2, BoNT/A3 andBoNT/A4; a H_(N) region from subtype BoNT/B1, BoNT/B2, BoNT/B bivalentand BoNT/B nonproteolytic; a H_(N) region from subtype BoNT/C1-1 andBoNT/C1-2; a H_(N) region from subtype BoNT/E1, BoNT/E2 and BoNT/E3; anda H_(N) region from subtype BoNT/F1, BoNT/F2, BoNT/F3 and BoNT/F4. AClostridial toxin H_(N) region subtype can function in substantially thesame manner as the reference Clostridial toxin H_(N) region on which theClostridial toxin H_(N) region subtype is based, and can be substitutedfor the reference Clostridial toxin H_(N) region in any aspect of thepresent invention.

As used herein, the term “non-naturally occurring Clostridial toxinH_(N) region variant” means any Clostridial toxin H_(N) region producedwith the aid of human manipulation, including, without limitation,Clostridial toxin H_(N) regions produced by genetic engineering usingrandom mutagenesis or rational design and Clostridial toxin H_(N)regions produced by chemical synthesis. Non-limiting examples ofnon-naturally occurring Clostridial toxin H_(N) region variants include,e.g., conservative Clostridial toxin H_(N) region variants,non-conservative Clostridial toxin H_(N) region variants, Clostridialtoxin H_(N) region chimeric variants and active Clostridial toxin H_(N)region fragments.

As used herein, the term “conservative Clostridial toxin H_(N) regionvariant” means a Clostridial toxin H_(N) region that has at least oneamino acid substituted by another amino acid or an amino acid analogthat has at least one property similar to that of the original aminoacid from the reference Clostridial toxin H_(N) region sequence (Table1). Examples of properties include, without limitation, similar size,topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservativeClostridial toxin H_(N) region variant can function in substantially thesame manner as the reference Clostridial toxin H_(N) region on which theconservative Clostridial toxin H_(N) region variant is based, and can besubstituted for the reference Clostridial toxin H_(N) region in anyaspect of the present invention. A conservative Clostridial toxin H_(N)region variant may substitute one or more amino acids, two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, ten or more amino acids, 20 or more amino acids, 30 or moreamino acids, 40 or more amino acids, 50 or more amino acids, 100 or moreamino acids or 200 or more amino acids from the reference Clostridialtoxin H_(N) region on which the conservative Clostridial toxin H_(N)region variant is based. A conservative Clostridial toxin H_(N) regionvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference Clostridial toxinH_(N) region on which the conservative Clostridial toxin H_(N) regionvariant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial toxin H_(N) regionon which the conservative Clostridial toxin H_(N) region variant isbased. Non-limiting examples of a conservative Clostridial toxin H_(N)region variant include, e.g., conservative BoNT/A H_(N) region variants,conservative BoNT/B H_(N) region variants, conservative BoNT/C1 H_(N)region variants, conservative BoNT/D H_(N) region variants, conservativeBoNT/E H_(N) region variants, conservative BoNT/F H_(N) region variants,conservative BoNT/G H_(N) region variants, and conservative TeNT H_(N)region variants.

As used herein, the term “non-conservative Clostridial toxin H_(N)region variant” means a Clostridial toxin H_(N) region in which 1) atleast one amino acid is deleted from the reference Clostridial toxinH_(N) region on which the non-conservative Clostridial toxin H_(N)region variant is based; 2) at least one amino acid added to thereference Clostridial toxin H_(N) region on which the non-conservativeClostridial toxin H_(N) region is based; or 3) at least one amino acidis substituted by another amino acid or an amino acid analog that doesnot share any property similar to that of the original amino acid fromthe reference Clostridial toxin H_(N) region sequence (Table 1). Anon-conservative Clostridial toxin H_(N) region variant can function insubstantially the same manner as the reference Clostridial toxin H_(N)region on which the non-conservative Clostridial toxin H_(N) regionvariant is based, and can be substituted for the reference Clostridialtoxin H_(N) region in any aspect of the present invention. Anon-conservative Clostridial toxin H_(N) region variant can delete oneor more amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, and ten or moreamino acids from the reference Clostridial toxin H_(N) region on whichthe non-conservative Clostridial toxin H_(N) region variant is based. Anon-conservative Clostridial toxin H_(N) region variant can add one ormore amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, and ten or moreamino acids to the reference Clostridial toxin H_(N) region on which thenon-conservative Clostridial toxin H_(N) region variant is based. Anon-conservative Clostridial toxin H_(N) region variant may substituteone or more amino acids, two or more amino acids, three or more aminoacids, four or more amino acids, five or more amino acids, ten or moreamino acids, 20 or more amino acids, 30 or more amino acids, 40 or moreamino acids, 50 or more amino acids, 100 or more amino acids or 200 ormore amino acids from the reference Clostridial toxin H_(N) region onwhich the non-conservative Clostridial toxin H_(N) region variant isbased. A non-conservative Clostridial toxin H_(N) region variant canalso substitute at least 10 contiguous amino acids, at least 15contiguous amino acids, at least 20 contiguous amino acids, or at least25 contiguous amino acids from the reference Clostridial toxin H_(N)region on which the non-conservative Clostridial toxin H_(N) regionvariant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial toxin H_(N) regionon which the non-conservative Clostridial toxin H_(N) region variant isbased. Non-limiting examples of a non-conservative Clostridial toxinH_(N) region variant include, e.g., non-conservative BoNT/A H_(N) regionvariants, non-conservative BoNT/B H_(N) region variants,non-conservative BoNT/C1 H_(N) region variants, non-conservative BoNT/DH_(N) region variants, non-conservative BoNT/E H_(N) region variants,non-conservative BoNT/F H_(N) region variants, non-conservative BoNT/GH_(N) region variants, and non-conservative TeNT H_(N) region variants.

As used herein, the term “Clostridial toxin H_(N) region chimeric” meansa polypeptide comprising at least a portion of a Clostridial toxin H_(N)region and at least a portion of at least one other polypeptide to forma toxin H_(N) region with at least one property different from thereference Clostridial toxin H_(N) regions of Table 1, with the provisothat this Clostridial toxin H_(N) region chimeric is still capable offacilitating the release of the LC from intracellular vesicles into thecytoplasm of the target cell and thus participate in executing theoverall cellular mechanism whereby a Clostridial toxin proteolyticallycleaves a substrate.

As used herein, the term “active Clostridial toxin H_(N) regionfragment” means any of a variety of Clostridial toxin fragmentscomprising the H_(N) region can be useful in aspects of the presentinvention with the proviso that these active fragments can facilitatethe release of the LC from intracellular vesicles into the cytoplasm ofthe target cell and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. The H_(N) regions from the heavy chains of Clostridial toxinsare approximately 410-430 amino acids in length and comprise atranslocation domain (Table 1). Research has shown that the entirelength of a H_(N) region from a Clostridial toxin heavy chain is notnecessary for the translocating activity of the translocation domain.Thus, aspects of this embodiment can include Clostridial toxin H_(N)regions comprising a translocation domain having a length of, e.g., atleast 350 amino acids, at least 375 amino acids, at least 400 aminoacids and at least 425 amino acids. Other aspects of this embodiment caninclude Clostridial toxin H_(N) regions comprising translocation domainhaving a length of, e.g., at most 350 amino acids, at most 375 aminoacids, at most 400 amino acids and at most 425 amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin H_(N) regionvariants and non-naturally-occurring Clostridial toxin H_(N) regionvariants, including, without limitation, global methods, local methodsand hybrid methods, such as, e.g., segment approach methods. Protocolsto determine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin translocationdomain. In an aspect of this embodiment, a Clostridial toxintranslocation domain comprises a naturally occurring Clostridial toxinH_(N) region variant, such as, e.g., a Clostridial toxin H_(N) regionisoform or a Clostridial toxin H_(N) region subtype. In another aspectof this embodiment, a Clostridial toxin translocation domain comprises anon-naturally occurring Clostridial toxin H_(N) region variant, such as,e.g., a conservative Clostridial toxin H_(N) region variant, anon-conservative Clostridial toxin H_(N) region variant, a Clostridialtoxin chimeric H_(N) region, an active Clostridial toxin H_(N) regionfragment, or any combination thereof.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/A H_(N) region. In an aspect of this embodiment, aBoNT/A H_(N) region comprises amino acids 449-873 of SEQ ID NO: 1. Inanother aspect of this embodiment, a BoNT/A H_(N) region comprises anaturally occurring BoNT/A H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/A isoform or a H_(N) region from a BoNT/A subtype. Inanother aspect of this embodiment, a BoNT/A H_(N) region comprises aminoacids 449-873 of a naturally occurring BoNT/A H_(N) region variant ofSEQ ID NO: 1, such as, e.g., amino acids 449-873 of a BoNT/A isoform ofSEQ ID NO: 1 or amino acids 449-873 of a BoNT/A subtype of SEQ ID NO: 1.In still another aspect of this embodiment, a BoNT/A H_(N) regioncomprises a non-naturally occurring BoNT/A H_(N) region variant, suchas, e.g., a conservative BoNT/A H_(N) region variant, a non-conservativeBoNT/A H_(N) region variant, a BoNT/A chimeric H_(N) region, an activeBoNT/A H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/A H_(N) region comprises aminoacids 449-873 of a non-naturally occurring BoNT/A H_(N) region variantof SEQ ID NO: 1, such as, e.g., amino acids 449-873 of a conservativeBoNT/A H_(N) region variant of SEQ ID NO: 1, amino acids 449-873 of anon-conservative BoNT/A H_(N) region variant of SEQ ID NO: 1, aminoacids 449-873 of an active BoNT/A H_(N) region fragment of SEQ ID NO: 1,or any combination thereof.

In other aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 449-873 of SEQ ID NO: 1, at least 75% amino acid identity withamino acids 449-873 of SEQ ID NO: 1, at least 80% amino acid identitywith amino acids 449-873 of SEQ ID NO: 1, at least 85% amino acididentity with amino acids 449-873 of SEQ ID NO: 1, at least 90% aminoacid identity with amino acids 449-873 of SEQ ID NO: 1 or at least 95%amino acid identity with amino acids 449-873 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 449-873 of SEQ ID NO: 1, at most 75% amino acid identity withamino acids 449-873 of SEQ ID NO: 1, at most 80% amino acid identitywith amino acids 449-873 of SEQ ID NO: 1, at most 85% amino acididentity with amino acids 449-873 of SEQ ID NO: 1, at most 90% aminoacid identity with amino acids 449-873 of SEQ ID NO: 1 or at most 95%amino acid identity with amino acids 449-873 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 449-873 of SEQ ID NO: 1. Inother aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 449-873 of SEQ ID NO: 1. Inyet other aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 449-873 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 449-873 of SEQ ID NO: 1. In stillother aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 449-873 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 449-873 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 449-873 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 449-873 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 449-873 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 449-873 of SEQ ID NO: 1. In stillother aspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 449-873 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 449-873 of SEQ ID NO: 1.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/B H_(N) region. In an aspect of this embodiment, aBoNT/B H_(N) region comprises amino acids 442-860 of SEQ ID NO: 2. Inanother aspect of this embodiment, a BoNT/B H_(N) region comprises anaturally occurring BoNT/B H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/B isoform or a H_(N) region from a BoNT/B subtype. Inanother aspect of this embodiment, a BoNT/B H_(N) region comprises aminoacids 442-860 of a naturally occurring BoNT/B H_(N) region variant ofSEQ ID NO: 2, such as, e.g., amino acids 442-860 of a BoNT/B isoform ofSEQ ID NO: 2 or amino acids 442-860 of a BoNT/B subtype of SEQ ID NO: 2.In still another aspect of this embodiment, a BoNT/B H_(N) regioncomprises a non-naturally occurring BoNT/B H_(N) region variant, suchas, e.g., a conservative BoNT/B H_(N) region variant, a non-conservativeBoNT/B H_(N) region variant, a BoNT/B chimeric H_(N) region, an activeBoNT/B H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/B H_(N) region comprises aminoacids 442-860 of a non-naturally occurring BoNT/B H_(N) region variantof SEQ ID NO: 2, such as, e.g., amino acids 442-860 of a conservativeBoNT/B H_(N) region variant of SEQ ID NO: 2, amino acids 442-860 of anon-conservative BoNT/B H_(N) region variant of SEQ ID NO: 2, aminoacids 442-860 of an active BoNT/B H_(N) region fragment of SEQ ID NO: 2,or any combination thereof.

In other aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 442-860 of SEQ ID NO: 2, at least 75% amino acid identity withamino acids 442-860 of SEQ ID NO: 2, at least 80% amino acid identitywith amino acids 442-860 of SEQ ID NO: 2, at least 85% amino acididentity with amino acids 442-860 of SEQ ID NO: 2, at least 90% aminoacid identity with amino acids 442-860 of SEQ ID NO: 2 or at least 95%amino acid identity with amino acids 442-860 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 442-860 of SEQ ID NO: 2, at most 75% amino acid identity withamino acids 442-860 of SEQ ID NO: 2, at most 80% amino acid identitywith amino acids 442-860 of SEQ ID NO: 2, at most 85% amino acididentity with amino acids 442-860 of SEQ ID NO: 2, at most 90% aminoacid identity with amino acids 442-860 of SEQ ID NO: 2 or at most 95%amino acid identity with amino acids 442-860 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 442-860 of SEQ ID NO: 2. Inother aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 442-860 of SEQ ID NO: 2. Inyet other aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 442-860 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 442-860 of SEQ ID NO: 2. In stillother aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 442-860 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 442-860 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 442-860 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 442-860 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 442-860 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 442-860 of SEQ ID NO: 2. In stillother aspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 442-860 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 442-860 of SEQ ID NO: 2.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/C1 H_(N) region. In an aspect of this embodiment, aBoNT/C1 H_(N) region comprises amino acids 450-868 of SEQ ID NO: 3. Inanother aspect of this embodiment, a BoNT/C1 H_(N) region comprises anaturally occurring BoNT/C1 H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/C1 isoform or a H_(N) region from a BoNT/C1 subtype.In another aspect of this embodiment, a BoNT/C1 H_(N) region comprisesamino acids 450-868 of a naturally occurring BoNT/C1 H_(N) regionvariant of SEQ ID NO: 3, such as, e.g., amino acids 450-868 of a BoNT/C1isoform of SEQ ID NO: 3 or amino acids 450-868 of a BoNT/C1 subtype ofSEQ ID NO: 3. In still another aspect of this embodiment, a BoNT/C1H_(N) region comprises a non-naturally occurring BoNT/C1 H_(N) regionvariant, such as, e.g., a conservative BoNT/C1 H_(N) region variant, anon-conservative BoNT/C1 H_(N) region variant, a BoNT/C1 chimeric H_(N)region, an active BoNT/C1 H_(N) region fragment, or any combinationthereof. In still another aspect of this embodiment, a BoNT/C1 H_(N)region comprises amino acids 450-868 of a non-naturally occurringBoNT/C1 H_(N) region variant of SEQ ID NO: 3, such as, e.g., amino acids450-868 of a conservative BoNT/C1 H_(N) region variant of SEQ ID NO: 3,amino acids 450-868 of a non-conservative BoNT/C1 H_(N) region variantof SEQ ID NO: 3, amino acids 450-868 of an active BoNT/C1 H_(N) regionfragment of SEQ ID NO: 3, or any combination thereof.

In other aspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 450-868 of SEQ ID NO: 3, at least 75% amino acid identity withamino acids 450-868 of SEQ ID NO: 3, at least 80% amino acid identitywith amino acids 450-868 of SEQ ID NO: 3, at least 85% amino acididentity with amino acids 450-868 of SEQ ID NO: 3, at least 90% aminoacid identity with amino acids 450-868 of SEQ ID NO: 3 or at least 95%amino acid identity with amino acids 450-868 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 450-868 of SEQ ID NO: 3, at most 75% amino acid identity withamino acids 450-868 of SEQ ID NO: 3, at most 80% amino acid identitywith amino acids 450-868 of SEQ ID NO: 3, at most 85% amino acididentity with amino acids 450-868 of SEQ ID NO: 3, at most 90% aminoacid identity with amino acids 450-868 of SEQ ID NO: 3 or at most 95%amino acid identity with amino acids 450-868 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 450-868 of SEQ ID NO: 3. Inother aspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 450-868 of SEQ ID NO: 3. Inyet other aspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 450-868 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 450-868 of SEQ ID NO: 3. In stillother aspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 450-868 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 450-868 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 450-868 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 450-868 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 450-868 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 450-868 of SEQ ID NO: 3. In stillother aspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 450-868 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 450-868 of SEQ ID NO: 3.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/D H_(N) region. In an aspect of this embodiment, aBoNT/D H_(N) region comprises amino acids 446-864 of SEQ ID NO: 4. Inanother aspect of this embodiment, a BoNT/D H_(N) region comprises anaturally occurring BoNT/D H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/D isoform or a H_(N) region from a BoNT/D subtype. Inanother aspect of this embodiment, a BoNT/D H_(N) region comprises aminoacids 446-864 of a naturally occurring BoNT/D H_(N) region variant ofSEQ ID NO: 4, such as, e.g., amino acids 446-864 of a BoNT/D isoform ofSEQ ID NO: 4 or amino acids 446-864 of a BoNT/D subtype of SEQ ID NO: 4.In still another aspect of this embodiment, a BoNT/D H_(N) regioncomprises a non-naturally occurring BoNT/D H_(N) region variant, suchas, e.g., a conservative BoNT/D H_(N) region variant, a non-conservativeBoNT/D H_(N) region variant, a BoNT/D chimeric H_(N) region, an activeBoNT/D H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/D H_(N) region comprises aminoacids 446-864 of a non-naturally occurring BoNT/D H_(N) region variantof SEQ ID NO: 4, such as, e.g., amino acids 446-864 of a conservativeBoNT/D H_(N) region variant of SEQ ID NO: 4, amino acids 446-864 of anon-conservative BoNT/D H_(N) region variant of SEQ ID NO: 4, aminoacids 446-864 of an active BoNT/D H_(N) region fragment of SEQ ID NO: 4,or any combination thereof.

In other aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 446-864 of SEQ ID NO: 4, at least 75% amino acid identity withamino acids 446-864 of SEQ ID NO: 4, at least 80% amino acid identitywith amino acids 446-864 of SEQ ID NO: 4, at least 85% amino acididentity with amino acids 446-864 of SEQ ID NO: 4, at least 90% aminoacid identity with amino acids 446-864 of SEQ ID NO: 4 or at least 95%amino acid identity with amino acids 446-864 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 446-864 of SEQ ID NO: 4, at most 75% amino acid identity withamino acids 446-864 of SEQ ID NO: 4, at most 80% amino acid identitywith amino acids 446-864 of SEQ ID NO: 4, at most 85% amino acididentity with amino acids 446-864 of SEQ ID NO: 4, at most 90% aminoacid identity with amino acids 446-864 of SEQ ID NO: 4 or at most 95%amino acid identity with amino acids 446-864 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 446-864 of SEQ ID NO: 4. Inother aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 446-864 of SEQ ID NO: 4. Inyet other aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 446-864 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 446-864 of SEQ ID NO: 4. In stillother aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 446-864 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 446-864 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 446-864 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 446-864 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 446-864 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 446-864 of SEQ ID NO: 4. In stillother aspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 446-864 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 446-864 of SEQ ID NO: 4.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/E H_(N) region. In an aspect of this embodiment, aBoNT/E H_(N) region comprises amino acids 423-847 of SEQ ID NO: 5. Inanother aspect of this embodiment, a BoNT/E H_(N) region comprises anaturally occurring BoNT/E H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/E isoform or a H_(N) region from a BoNT/E subtype. Inanother aspect of this embodiment, a BoNT/E H_(N) region comprises aminoacids 423-847 of a naturally occurring BoNT/E H_(N) region variant ofSEQ ID NO: 5, such as, e.g., amino acids 423-847 of a BoNT/E isoform ofSEQ ID NO: 5 or amino acids 423-847 of a BoNT/E subtype of SEQ ID NO: 5.In still another aspect of this embodiment, a BoNT/E H_(N) regioncomprises a non-naturally occurring BoNT/E H_(N) region variant, suchas, e.g., a conservative BoNT/E H_(N) region variant, a non-conservativeBoNT/E H_(N) region variant, a BoNT/E chimeric H_(N) region, an activeBoNT/E H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/E H_(N) region comprises aminoacids 423-847 of a non-naturally occurring BoNT/E H_(N) region variantof SEQ ID NO: 5, such as, e.g., amino acids 423-847 of a conservativeBoNT/E H_(N) region variant of SEQ ID NO: 5, amino acids 423-847 of anon-conservative BoNT/E H_(N) region variant of SEQ ID NO: 5, aminoacids 423-847 of an active BoNT/E H_(N) region fragment of SEQ ID NO: 5,or any combination thereof.

In other aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 423-847 of SEQ ID NO: 5, at least 75% amino acid identity withamino acids 423-847 of SEQ ID NO: 5, at least 80% amino acid identitywith amino acids 423-847 of SEQ ID NO: 5, at least 85% amino acididentity with amino acids 423-847 of SEQ ID NO: 5, at least 90% aminoacid identity with amino acids 423-847 of SEQ ID NO: 5 or at least 95%amino acid identity with amino acids 423-847 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 423-847 of SEQ ID NO: 5, at most 75% amino acid identity withamino acids 423-847 of SEQ ID NO: 5, at most 80% amino acid identitywith amino acids 423-847 of SEQ ID NO: 5, at most 85% amino acididentity with amino acids 423-847 of SEQ ID NO: 5, at most 90% aminoacid identity with amino acids 423-847 of SEQ ID NO: 5 or at most 95%amino acid identity with amino acids 423-847 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 423-847 of SEQ ID NO: 5. Inother aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 423-847 of SEQ ID NO: 5. Inyet other aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 423-847 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 423-847 of SEQ ID NO: 5. In stillother aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 423-847 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 423-847 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 423-847 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 423-847 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 423-847 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 423-847 of SEQ ID NO: 5. In stillother aspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 423-847 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 423-847 of SEQ ID NO: 5.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/F H_(N) region. In an aspect of this embodiment, aBoNT/F H_(N) region comprises amino acids 440-866 of SEQ ID NO: 6. Inanother aspect of this embodiment, a BoNT/F H_(N) region comprises anaturally occurring BoNT/F H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/F isoform or a H_(N) region from a BoNT/F subtype. Inanother aspect of this embodiment, a BoNT/F H_(N) region comprises aminoacids 440-866 of a naturally occurring BoNT/F H_(N) region variant ofSEQ ID NO: 6, such as, e.g., amino acids 440-866 of a BoNT/F isoform ofSEQ ID NO: 6 or amino acids 440-866 of a BoNT/F subtype of SEQ ID NO: 6.In still another aspect of this embodiment, a BoNT/F H_(N) regioncomprises a non-naturally occurring BoNT/F H_(N) region variant, suchas, e.g., a conservative BoNT/F H_(N) region variant, a non-conservativeBoNT/F H_(N) region variant, a BoNT/F chimeric H_(N) region, an activeBoNT/F H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/F H_(N) region comprises aminoacids 440-866 of a non-naturally occurring BoNT/F H_(N) region variantof SEQ ID NO: 6, such as, e.g., amino acids 440-866 of a conservativeBoNT/F H_(N) region variant of SEQ ID NO: 6, amino acids 440-866 of anon-conservative BoNT/F H_(N) region variant of SEQ ID NO: 6, aminoacids 440-866 of an active BoNT/F H_(N) region fragment of SEQ ID NO: 6,or any combination thereof.

In other aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 440-866 of SEQ ID NO: 6, at least 75% amino acid identity withamino acids 440-866 of SEQ ID NO: 6, at least 80% amino acid identitywith amino acids 440-866 of SEQ ID NO: 6, at least 85% amino acididentity with amino acids 440-866 of SEQ ID NO: 6, at least 90% aminoacid identity with amino acids 440-866 of SEQ ID NO: 6 or at least 95%amino acid identity with amino acids 440-866 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 440-866 of SEQ ID NO: 6, at most 75% amino acid identity withamino acids 440-866 of SEQ ID NO: 6, at most 80% amino acid identitywith amino acids 440-866 of SEQ ID NO: 6, at most 85% amino acididentity with amino acids 440-866 of SEQ ID NO: 6, at most 90% aminoacid identity with amino acids 440-866 of SEQ ID NO: 6 or at most 95%amino acid identity with amino acids 440-866 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 440-866 of SEQ ID NO: 6. Inother aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 440-866 of SEQ ID NO: 6. Inyet other aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 440-866 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 440-866 of SEQ ID NO: 6. In stillother aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 440-866 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 440-866 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 440-866 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 440-866 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 440-866 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 440-866 of SEQ ID NO: 6. In stillother aspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 440-866 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 440-866 of SEQ ID NO: 6.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/G H_(N) region. In an aspect of this embodiment, aBoNT/G H_(N) region comprises amino acids 447-865 of SEQ ID NO: 7. Inanother aspect of this embodiment, a BoNT/G H_(N) region comprises anaturally occurring BoNT/G H_(N) region variant, such as, e.g., a H_(N)region from a BoNT/G isoform or a H_(N) region from a BoNT/G subtype. Inanother aspect of this embodiment, a BoNT/G H_(N) region comprises aminoacids 447-865 of a naturally occurring BoNT/G H_(N) region variant ofSEQ ID NO: 7, such as, e.g., amino acids 447-865 of a BoNT/G isoform ofSEQ ID NO: 7 or amino acids 447-865 of a BoNT/G subtype of SEQ ID NO: 7.In still another aspect of this embodiment, a BoNT/G H_(N) regioncomprises a non-naturally occurring BoNT/G H_(N) region variant, suchas, e.g., a conservative BoNT/G H_(N) region variant, a non-conservativeBoNT/G H_(N) region variant, a BoNT/G chimeric H_(N) region, an activeBoNT/G H_(N) region fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/G H_(N) region comprises aminoacids 447-865 of a non-naturally occurring BoNT/G H_(N) region variantof SEQ ID NO: 7, such as, e.g., amino acids 447-865 of a conservativeBoNT/G H_(N) region variant of SEQ ID NO: 7, amino acids 447-865 of anon-conservative BoNT/G H_(N) region variant of SEQ ID NO: 7, aminoacids 447-865 of an active BoNT/G H_(N) region fragment of SEQ ID NO: 7,or any combination thereof.

In other aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 447-865 of SEQ ID NO: 7, at least 75% amino acid identity withamino acids 447-865 of SEQ ID NO: 7, at least 80% amino acid identitywith amino acids 447-865 of SEQ ID NO: 7, at least 85% amino acididentity with amino acids 447-865 of SEQ ID NO: 7, at least 90% aminoacid identity with amino acids 447-865 of SEQ ID NO: 7 or at least 95%amino acid identity with amino acids 447-865 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 447-865 of SEQ ID NO: 7, at most 75% amino acid identity withamino acids 447-865 of SEQ ID NO: 7, at most 80% amino acid identitywith amino acids 447-865 of SEQ ID NO: 7, at most 85% amino acididentity with amino acids 447-865 of SEQ ID NO: 7, at most 90% aminoacid identity with amino acids 447-865 of SEQ ID NO: 7 or at most 95%amino acid identity with amino acids 447-865 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 447-865 of SEQ ID NO: 7. Inother aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 447-865 of SEQ ID NO: 7. Inyet other aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 447-865 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 447-865 of SEQ ID NO: 7. In stillother aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 447-865 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid additions relative to amino acids 447-865 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 447-865 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 447-865 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 447-865 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino aciddeletions relative to amino acids 447-865 of SEQ ID NO: 7. In stillother aspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 447-865 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidadditions relative to amino acids 447-865 of SEQ ID NO: 7.

In another embodiment, a Clostridial toxin translocation domaincomprises a TeNT H_(N) region. In an aspect of this embodiment, a TeNTH_(N) region comprises amino acids 458-881 of SEQ ID NO: 8. In anotheraspect of this embodiment, a TeNT H_(N) region comprises a naturallyoccurring TeNT H_(N) region variant, such as, e.g., a H_(N) region froma TeNT isoform or a H_(N) region from a TeNT subtype. In another aspectof this embodiment, a TeNT H_(N) region comprises amino acids 458-881 ofa naturally occurring TeNT H_(N) region variant of SEQ ID NO: 8, suchas, e.g., amino acids 458-881 of a TeNT isoform of SEQ ID NO: 8 or aminoacids 458-881 of a TeNT subtype of SEQ ID NO: 8. In still another aspectof this embodiment, a TeNT H_(N) region comprises a non-naturallyoccurring TeNT H_(N) region variant, such as, e.g., a conservative TeNTH_(N) region variant, a non-conservative TeNT H_(N) region variant, aTeNT chimeric H_(N) region, an active TeNT H_(N) region fragment, or anycombination thereof. In still another aspect of this embodiment, a TeNTH_(N) region comprises amino acids 458-881 of a non-naturally occurringTeNT H_(N) region variant of SEQ ID NO: 8, such as, e.g., amino acids458-881 of a conservative TeNT H_(N) region variant of SEQ ID NO: 8,amino acids 458-881 of a non-conservative TeNT H_(N) region variant ofSEQ ID NO: 8, amino acids 458-881 of an active TeNT H_(N) regionfragment of SEQ ID NO: 8, or any combination thereof.

In other aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 458-881 of SEQ ID NO: 8, at least 75% amino acid identity withamino acids 458-881 of SEQ ID NO: 8, at least 80% amino acid identitywith amino acids 458-881 of SEQ ID NO: 8, at least 85% amino acididentity with amino acids 458-881 of SEQ ID NO: 8, at least 90% aminoacid identity with amino acids 458-881 of SEQ ID NO: 8 or at least 95%amino acid identity with amino acids 458-881 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 458-881 of SEQ ID NO: 8, at most 75% amino acid identity withamino acids 458-881 of SEQ ID NO: 8, at most 80% amino acid identitywith amino acids 458-881 of SEQ ID NO: 8, at most 85% amino acididentity with amino acids 458-881 of SEQ ID NO: 8, at most 90% aminoacid identity with amino acids 458-881 of SEQ ID NO: 8 or at most 95%amino acid identity with amino acids 458-881 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 458-881 of SEQ ID NO: 8. Inother aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid substitutions relative to amino acids 458-881 of SEQ ID NO: 8. Inyet other aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous aminoacid deletions relative to amino acids 458-881 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT H_(N) region comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid deletionsrelative to amino acids 458-881 of SEQ ID NO: 8. In still other aspectsof this embodiment, a TeNT H_(N) region comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 non-contiguous amino acid additions relativeto amino acids 458-881 of SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT H_(N) region comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 non-contiguous amino acid additions relative to aminoacids 458-881 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(N) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acidsubstitutions relative to amino acids 458-881 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT H_(N) region comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutionsrelative to amino acids 458-881 of SEQ ID NO: 8. In yet other aspects ofthis embodiment, a TeNT H_(N) region comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100 or 200 contiguous amino acid deletions relative toamino acids 458-881 of SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT H_(N) region comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid deletions relative to aminoacids 458-881 of SEQ ID NO: 8. In still other aspects of thisembodiment, a TeNT H_(N) region comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,40, 50, 100 or 200 contiguous amino acid additions relative to aminoacids 458-881 of SEQ ID NO: 8. In other aspects of this embodiment, aTeNT H_(N) region comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100or 200 contiguous amino acid additions relative to amino acids 458-881of SEQ ID NO: 8.

Aspects of the present invention provide, in part, a translocationfacilitating domain. As used herein, the term “translocationfacilitating domain” means any polypeptide that can further facilitatethe translocation step of the intoxication process that mediatesClostridial toxin light chain translocation. Thus, a translocationfacilitating domain assists the Clostridial toxin translocation domainin the movement of a Clostridial toxin light chain across a membrane andencompasses the movement of a Clostridial toxin light chain through themembrane of an intracellular vesicle into the cytoplasm of a cell. Anon-limiting example of a translocation facilitating domain is aClostridial toxin translocation facilitating domain, such as, e.g., aClostridial toxin H_(CN) region such as, e.g., a BoNT/A H_(CN) region, aBoNT/B H_(CN) region, a BoNT/C1 H_(CN) region, a BoNT/D H_(CN) region, aBoNT/E H_(CN) region, a BoNT/F H_(CN) region, a BoNT/G H_(CN) region,and a TeNT H_(CN) region. Another non-limiting example of atranslocation facilitating domain is a viral fusogenic peptide domainfound in an enveloped virus, such as, e.g., an influenzavirus, analphavirus, a vesiculovirus, a respirovirus, a morbillivirus, anavulavirus, a henipavirus, a metapneumovirus and a foamy virus.

Thus, in an embodiment, a translocation facilitating domain facilitatesthe Clostridial toxin translocation domain in the movement of aClostridial toxin light chain across a membrane. In aspects of thisembodiment, a translocation facilitating domain facilitates theClostridial toxin translocation domain in the movement of a Clostridialtoxin light chain across a membrane by increasing the amount ofClostridial toxin light chain in the cytoplasm by, e.g., at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or at least 100%. In other aspectsof this embodiment, a translocation facilitating domain facilitates theClostridial toxin translocation domain in the movement of a Clostridialtoxin light chain across a membrane by increasing the amount ofClostridial toxin light chain in the cytoplasm by, e.g., at leasttwo-fold, at least three-fold, at least four-fold, at least five-fold,at least ten-fold or at least twenty-fold. In yet other aspects of thisembodiment, a translocation facilitating domain facilitates theClostridial toxin translocation domain in the movement of a Clostridialtoxin light chain across a membrane by increasing the amount ofClostridial toxin light chain in the cytoplasm by, e.g., at most 10%, atmost 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most70%, at most 80%, at most 90% or at most 100%. In other aspects of thisembodiment, a translocation facilitating domain facilitates theClostridial toxin translocation domain in the movement of a Clostridialtoxin light chain across a membrane by increasing the amount ofClostridial toxin light chain in the cytoplasm by, e.g., at mosttwo-fold, at most three-fold, at most four-fold, at most five-fold, atmost ten-fold or at most twenty-fold.

A Clostridial toxin “translocation facilitating domain includes, withoutlimitation, naturally occurring Clostridial toxin H_(CN) regionvariants, such as, e.g., Clostridial toxin H_(CN) region isoforms andClostridial toxin H_(CN) region subtypes; non-naturally occurringClostridial toxin H_(CN) region variants, such as, e.g., conservativeClostridial toxin H_(CN) region variants, non-conservative Clostridialtoxin H_(CN) region variants, Clostridial toxin H_(CN) region chimerics,active Clostridial toxin H_(CN) region fragments thereof, or anycombination thereof.

As used herein, the term “Clostridial toxin H_(CN) region variant,”whether naturally-occurring or non-naturally-occurring, means aClostridial toxin H_(CN) region that has at least one amino acid changefrom the corresponding region of the disclosed reference sequences (seeTable 1) and can be described in percent identity to the correspondingregion of that reference sequence. Unless expressly indicated, allClostridial toxin H_(CN) region variants disclosed in the presentspecification are capable of further facilitating the translocation stepof the intoxication process that mediates Clostridial toxin light chaintranslocation. As non-limiting examples, a BoNT/A H_(CN) region variantcomprising amino acids 874-1110 of SEQ ID NO: 1 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 874-1110 ofSEQ ID NO: 1; a BoNT/B H_(CN) region variant comprising amino acids861-1097 of SEQ ID NO: 2 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 861-1097 of SEQ ID NO: 2; a BoNT/C1H_(CN) region variant comprising amino acids 869-1111 of SEQ ID NO: 3will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 869-1111 of SEQ ID NO: 3; a BoNT/D H_(CN) region variantcomprising amino acids 865-1098 of SEQ ID NO: 4 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 865-1098 ofSEQ ID NO: 4; a BoNT/E H_(CN) region variant comprising amino acids848-1085 of SEQ ID NO: 5 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 848-1085 of SEQ ID NO: 5; a BoNT/FH_(CN) region variant comprising amino acids 867-1105 of SEQ ID NO: 6will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 867-1105 of SEQ ID NO: 6; a BoNT/G H_(CN) region variantcomprising amino acids 866-1105 of SEQ ID NO: 7 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 866-1105 ofSEQ ID NO: 7; and a TeNT H_(CN) region variant comprising amino acids882-1127 of SEQ ID NO: 8 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 882-1127 of SEQ ID NO: 8.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxinH_(CN) region variants that differ somewhat in their amino acidsequence, and also in the nucleic acids encoding these proteins. Forexample, there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2,BoNT/A3 and BoNT/A4, with specific H_(CN) region subtypes showingapproximately 87% amino acid identity when compared to another BoNT/AH_(CN) region subtype. As used herein, the term “naturally occurringClostridial toxin H_(CN) region variant” means any Clostridial toxinH_(CN) region produced by a naturally-occurring process, including,without limitation, Clostridial toxin H_(CN) region isoforms producedfrom alternatively-spliced transcripts, Clostridial toxin H_(CN) regionisoforms produced by spontaneous mutation and Clostridial toxin H_(CN)region subtypes. A naturally occurring Clostridial toxin H_(CN) regionvariant can function in substantially the same manner as the referenceClostridial toxin H_(CN) region on which the naturally occurringClostridial toxin H_(CN) region variant is based, and can be substitutedfor the reference Clostridial toxin H_(CN) region in any aspect of thepresent invention. A naturally occurring Clostridial toxin H_(CN) regionvariant may substitute one or more amino acids, two or more amino acids,three or more amino acids, four or more amino acids, five or more aminoacids, ten or more amino acids, 20 or more amino acids, 30 or more aminoacids, 40 or more amino acids or 50 or more amino acids from thereference Clostridial toxin H_(CN) region on which the naturallyoccurring Clostridial toxin H_(CN) region variant is based. A naturallyoccurring Clostridial toxin H_(CN) region variant can also substitute atleast 10 contiguous amino acids, at least 15 contiguous amino acids, atleast 20 contiguous amino acids, or at least 25 contiguous amino acidsfrom the reference Clostridial toxin H_(CN) region on which thenaturally occurring Clostridial toxin H_(CN) region variant is based,that possess at least 50% amino acid identity, 65% amino acid identity,75% amino acid identity, 85% amino acid identity or 95% amino acididentity to the reference Clostridial toxin H_(CN) region on which thenaturally occurring Clostridial toxin H_(CN) region variant is based.

A non-limiting examples of a naturally occurring Clostridial toxinH_(CN) region variant is a Clostridial toxin H_(CN) region isoform suchas, e.g., a BoNT/A H_(CN) region isoform, a BoNT/B H_(CN) regionisoform, a BoNT/C1 H_(CN) region isoform, a BoNT/D H_(CN) regionisoform, a BoNT/E H_(CN) region isoform, a BoNT/F H_(CN) region isoform,a BoNT/G H_(CN) region isoform, and a TeNT H_(CN) region isoform. AClostridial toxin H_(CN) region isoform can function in substantiallythe same manner as the reference Clostridial toxin H_(CN) region onwhich the Clostridial toxin H_(CN) region isoform is based, and can besubstituted for the reference Clostridial toxin H_(CN) region in anyaspect of the present invention.

Another non-limiting examples of a naturally occurring Clostridial toxinH_(CN) region variant is a Clostridial toxin H_(CN) region subtype suchas, e.g., a H_(CN) region from subtype BoNT/A1, BoNT/A2, BoNT/A3 andBoNT/A4; a H_(CN) region from subtype BoNT/B1, BoNT/B2, BoNT/B bivalentand BoNT/B nonproteolytic; a H_(CN) region from subtype BoNT/C1-1 andBoNT/C1-2; a H_(CN) region from subtype BoNT/E1, BoNT/E2 and BoNT/E3;and a H_(CN) region from subtype BoNT/F1, BoNT/F2, BoNT/F3 and BoNT/F4.A Clostridial toxin H_(CN) region subtype can function in substantiallythe same manner as the reference Clostridial toxin H_(CN) region onwhich the Clostridial toxin H_(CN) region subtype is based, and can besubstituted for the reference Clostridial toxin H_(CN) region in anyaspect of the present invention.

As used herein, the term “non-naturally occurring Clostridial toxinH_(CN) region variant” means any Clostridial toxin H_(CN) regionproduced with the aid of human manipulation, including, withoutlimitation, Clostridial toxin H_(CN) regions produced by geneticengineering using random mutagenesis or rational design and Clostridialtoxin H_(CN) regions produced by chemical synthesis. Non-limitingexamples of non-naturally occurring Clostridial toxin H_(CN) regionvariants include, e.g., conservative Clostridial toxin H_(CN) regionvariants, non-conservative Clostridial toxin H_(CN) region variants,Clostridial toxin H_(CN) region chimeric variants and active Clostridialtoxin H_(CN) region fragments.

As used herein, the term “conservative Clostridial toxin H_(CN) regionvariant” means a Clostridial toxin H_(CN) region that has at least oneamino acid substituted by another amino acid or an amino acid analogthat has at least one property similar to that of the original aminoacid from the reference Clostridial toxin H_(CN) region sequence (Table1). Examples of properties include, without limitation, similar size,topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservativeClostridial toxin H_(CN) region variant can function in substantiallythe same manner as the reference Clostridial toxin H_(CN) region onwhich the conservative Clostridial toxin H_(CN) region variant is based,and can be substituted for the reference Clostridial toxin H_(CN) regionin any aspect of the present invention. A conservative Clostridial toxinH_(CN) region variant may substitute one or more amino acids, two ormore amino acids, three or more amino acids, four or more amino acids,five or more amino acids, ten or more amino acids, 20 or more aminoacids, 30 or more amino acids, 40 or more amino acids, 50 or more aminoacids or 100 or more amino acids from the reference Clostridial toxinH_(CN) region on which the conservative Clostridial toxin H_(CN) regionvariant is based. A conservative Clostridial toxin H_(CN) region variantcan also substitute at least 10 contiguous amino acids, at least 15contiguous amino acids, at least 20 contiguous amino acids, or at least25 contiguous amino acids from the reference Clostridial toxin H_(CN)region on which the conservative Clostridial toxin H_(CN) region variantis based, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference Clostridial toxin H_(CN) region on whichthe conservative Clostridial toxin H_(CN) region variant is based.Non-limiting examples of a conservative Clostridial toxin H_(CN) regionvariant include, e.g., conservative BoNT/A H_(CN) region variants,conservative BoNT/B H_(CN) region variants, conservative BoNT/C1 H_(CN)region variants, conservative BoNT/D H_(CN) region variants,conservative BoNT/E H_(CN) region variants, conservative BoNT/F H_(CN)region variants, conservative BoNT/G H_(CN) region variants, andconservative TeNT H_(CN) region variants.

As used herein, the term “non-conservative Clostridial toxin H_(CN)region variant” means a Clostridial toxin H_(CN) region in which 1) atleast one amino acid is deleted from the reference Clostridial toxinH_(CN) region on which the non-conservative Clostridial toxin H_(CN)region variant is based; 2) at least one amino acid added to thereference Clostridial toxin H_(CN) region on which the non-conservativeClostridial toxin H_(CN) region is based; or 3) at least one amino acidis substituted by another amino acid or an amino acid analog that doesnot share any property similar to that of the original amino acid fromthe reference Clostridial toxin H_(CN) region sequence (Table 1). Anon-conservative Clostridial toxin H_(CN) region variant can function insubstantially the same manner as the reference Clostridial toxin H_(CN)region on which the non-conservative Clostridial toxin H_(CN) regionvariant is based, and can be substituted for the reference Clostridialtoxin H_(CN) region in any aspect of the present invention. Anon-conservative Clostridial toxin H_(CN) region variant can delete oneor more amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, and ten or moreamino acids from the reference Clostridial toxin H_(CN) region on whichthe non-conservative Clostridial toxin H_(CN) region variant is based. Anon-conservative Clostridial toxin H_(CN) region variant can add one ormore amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids, and ten or moreamino acids to the reference Clostridial toxin H_(CN) region on whichthe non-conservative Clostridial toxin H_(CN) region variant is based. Anon-conservative Clostridial toxin H_(CN) region variant may substituteone or more amino acids, two or more amino acids, three or more aminoacids, four or more amino acids, five or more amino acids, ten or moreamino acids, 20 or more amino acids, 30 or more amino acids, 40 or moreamino acids, 50 or more amino acids or 100 or more amino acids from thereference Clostridial toxin H_(CN) region on which the non-conservativeClostridial toxin H_(CN) region variant is based. A non-conservativeClostridial toxin H_(CN) region variant can also substitute at least 10contiguous amino acids, at least 15 contiguous amino acids, at least 20contiguous amino acids, or at least 25 contiguous amino acids from thereference Clostridial toxin H_(CN) region on which the non-conservativeClostridial toxin H_(CN) region variant is based, that possess at least50% amino acid identity, 65% amino acid identity, 75% amino acididentity, 85% amino acid identity or 95% amino acid identity to thereference Clostridial toxin H_(CN) region on which the non-conservativeClostridial toxin H_(CN) region variant is based. Non-limiting examplesof a non-conservative Clostridial toxin H_(CN) region variant include,e.g., non-conservative BoNT/A H_(CN) region variants, non-conservativeBoNT/B H_(CN) region variants, non-conservative BoNT/C1 H_(CN) regionvariants, non-conservative BoNT/D H_(CN) region variants,non-conservative BoNT/E H_(CN) region variants, non-conservative BoNT/FH_(CN) region variants, non-conservative BoNT/G H_(CN) region variants,and non-conservative TeNT H_(CN) region variants.

As used herein, the term “Clostridial toxin H_(CN) region chimeric”means a polypeptide comprising at least a portion of a Clostridial toxinH_(CN) region and at least a portion of at least one other polypeptideto form a toxin H_(CN) region with at least one property different fromthe reference Clostridial toxin H_(CN) regions of Table 1, with theproviso that this Clostridial toxin H_(CN) region chimeric is stillcapable of further facilitating the translocation step of theintoxication process where the LC is released from intracellularvesicles into the cytoplasm of the target cell and thus participate inexecuting the overall cellular mechanism whereby a Clostridial toxinproteolytically cleaves a substrate.

As used herein, the term “active Clostridial toxin H_(CN) regionfragment” means any of a variety of Clostridial toxin fragmentscomprising the H_(CN) region can be useful in aspects of the presentinvention with the proviso that these active fragments can furtherfacilitate the translocation step of the intoxication process where theLC is released from intracellular vesicles into the cytoplasm of thetarget cell and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. The H_(CN) regions from the heavy chains of Clostridialtoxins are approximately 230-250 amino acids in length and comprise atranslocation domain (Table 1). Additionally, while a specific aminoacid positions have been identified to delineate the boundaries of theClostridial toxin H_(CN) region (Table 1), it is well known in the artthat the functional boundaries are not definitive. For example,amino-terminus of the H_(CN) domain for all naturally-occurringClostridial toxins is the H_(N) domain (translocation domain). Inexamining the structure for BoNT/A, a random coil linker region forms aboundary between the H_(N) and H_(CN) domains (see FIG. 7A). The BoNT/AH_(N) domain appears to end with an α-helix comprising amino acids N859to I873. Following the α-helix there is a random coil (I873 to 1878)that leads into the H_(CN) domain where a β-sheet begins at positionI878. The above residues define boundaries at the beginning or end ofdefined secondary structures and do not imply that there are notsignificant interactions (i.e., hydrophobic, H-bond, etc.) betweenresidues in the random coil region and one or both of the domains thatit links together. Thus, minimally an amino acid that defines theamino-terminal boundary of the BoNT/A H_(CN) domain comprises can be anyamino acid present in the amino acid region Y869 to L879. Similaranalysis indicates that minimally, the amino acid that defines theamino-terminal boundary of the BoNT/B H_(CN) domain can be any aminoacid present in the amino acid region Y856 to L866; the amino acid thatdefines the amino-terminal boundary of the BoNT/C1 H_(CN) domain can beany amino acid present in the amino acid region Y864 to L874; the aminoacid that defines the amino-terminal boundary of the BoNT/D H_(CN)domain can be any amino acid present in the amino acid region Y860 toL870; the amino acid that defines the amino-terminal boundary of theBoNT/E H_(CN) domain can be any amino acid present in the amino acidregion F843 to L853; the amino acid that defines the amino-terminalboundary of the BoNT/F H_(CN) domain can be any amino acid present inthe amino acid region L862 to L872; the amino acid that defines theamino-terminal boundary of the BoNT/G H_(CN) domain can be any aminoacid present in the amino acid region Y861 to L871; and the amino acidthat defines the amino-terminal boundary of the TeNT H_(CN) domain canbe any amino acid present in the amino acid region 1877 to L887.

Similarly, the carboxyl-terminal portion of the H_(CN) domain (i.e., thefusion point between the H_(CN) and H_(CC) domains) allnaturally-occurring Clostridial toxins comprises a range of amino acids.In defining the boundary of the BoNT/A H_(CN) domain as the beginning orthe end of ordered secondary structure, the H_(CN) domain could end atQ1091 of an α-helix and the H_(CC) domain could begin at K1109 of aβ-strand (see FIG. 7B). The intervening amino acid sequence betweenthese two domains comprises a longer random coil but, this does notimply that the random coil is not structurally important. In fact, thisrandom coil has a great deal of interaction with both the H_(CN) andH_(CC) domains (i.e., hydrophobic and H-bonding). Thus, minimally anamino acid that defines the carboxyl-terminal boundary of the BoNT/AH_(CN) domain comprises can be any amino acid present in the amino acidregion D1089 to Y1111. Similar analysis indicates that minimally, theamino acid that defines the carboxyl-terminal boundary of the BoNT/BH_(CN) domain can be any amino acid present in the amino acid regionK1076 to Y1098; the amino acid that defines the carboxyl-terminalboundary of the BoNT/C1 H_(CN) domain can be any amino acid present inthe amino acid region N1090 to Y1112; the amino acid that defines thecarboxyl-terminal boundary of the BoNT/D H_(CN) domain can be any aminoacid present in the amino acid region E1077 to Y1099; the amino acidthat defines the carboxyl-terminal boundary of the BoNT/E H_(CN) domaincan be any amino acid present in the amino acid region S1064 to Y1086;the amino acid that defines the carboxyl-terminal boundary of the BoNT/FH_(CN) domain can be any amino acid present in the amino acid regionS1084 to Y1106; the amino acid that defines the carboxyl-terminalboundary of the BoNT/G H_(CN) domain can be any amino acid present inthe amino acid region W1084 to Y1106; and the amino acid that definesthe carboxyl-terminal boundary of the TeNT H_(CN) domain can be anyamino acid present in the amino acid region T1106 to Y1128.

Thus, aspects of this embodiment can include Clostridial toxin H_(CN)regions comprising a translocation facilitating domain having a lengthof, e.g., at least 200 amino acids, at least 225 amino acids, at least250 amino acids and at least 275 amino acids. Other aspects of thisembodiment can include Clostridial toxin H_(CN) regions comprisingtranslocation facilitating domain having a length of, e.g., at most 200amino acids, at most 225 amino acids, at most 250 amino acids and atmost 275 amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin H_(CN) regionvariants and non-naturally-occurring Clostridial toxin H_(CN) regionvariants, including, without limitation, global methods, local methodsand hybrid methods, such as, e.g., segment approach methods. Protocolsto determine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin translocationdomain. In an aspect of this embodiment, a Clostridial toxintranslocation domain comprises a naturally occurring Clostridial toxinH_(CN) region variant, such as, e.g., a Clostridial toxin H_(CN) regionisoform or a Clostridial toxin H_(CN) region subtype. In another aspectof this embodiment, a Clostridial toxin translocation domain comprises anon-naturally occurring Clostridial toxin H_(CN) region variant, suchas, e.g., a conservative Clostridial toxin H_(CN) region variant, anon-conservative Clostridial toxin H_(CN) region variant, a Clostridialtoxin chimeric H_(CN) region, an active Clostridial toxin H_(CN) regionfragment, or any combination thereof.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/A H_(CN) region. In an aspect of this embodiment, aBoNT/A H_(CN) region comprises amino acids 874-1110 of SEQ ID NO: 1. Inanother aspect of this embodiment, a BoNT/A H_(CN) region comprises anaturally occurring BoNT/A H_(CN) region variant, such as, e.g., aH_(CN) region from a BoNT/A isoform or a H_(CN) region from a BoNT/Asubtype. In another aspect of this embodiment, a BoNT/A H_(CN) regioncomprises amino acids 874-1110 of a naturally occurring BoNT/A H_(CN)region variant of SEQ ID NO: 1, such as, e.g., amino acids 874-1110 of aBoNT/A isoform of SEQ ID NO: 1 or amino acids 874-1110 of a BoNT/Asubtype of SEQ ID NO: 1. In still another aspect of this embodiment, aBoNT/A H_(CN) region comprises a non-naturally occurring BoNT/A H_(CN)region variant, such as, e.g., a conservative BoNT/A H_(CN) regionvariant, a non-conservative BoNT/A H_(CN) region variant, a BoNT/Achimeric H_(CN) region, an active BoNT/A H_(CN) region fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/A H_(CN) region comprises amino acids 874-1110 of a non-naturallyoccurring BoNT/A H_(CN) region variant of SEQ ID NO: 1, such as, e.g.,amino acids 874-1110 of a conservative BoNT/A H_(CN) region variant ofSEQ ID NO: 1, amino acids 874-1110 of a non-conservative BoNT/A H_(CN)region variant of SEQ ID NO: 1, amino acids 874-1110 of an active BoNT/AH_(CN) region fragment of SEQ ID NO: 1, or any combination thereof.

In other aspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 874-1110 of SEQ ID NO: 1, at least 75% amino acid identity withamino acids 874-1110 of SEQ ID NO: 1, at least 80% amino acid identitywith amino acids 874-1110 of SEQ ID NO: 1, at least 85% amino acididentity with amino acids 874-1110 of SEQ ID NO: 1, at least 90% aminoacid identity with amino acids 874-1110 of SEQ ID NO: 1 or at least 95%amino acid identity with amino acids 874-1110 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 874-1110 of SEQ ID NO: 1, at most 75% amino acid identity withamino acids 874-1110 of SEQ ID NO: 1, at most 80% amino acid identitywith amino acids 874-1110 of SEQ ID NO: 1, at most 85% amino acididentity with amino acids 874-1110 of SEQ ID NO: 1, at most 90% aminoacid identity with amino acids 874-1110 of SEQ ID NO: 1 or at most 95%amino acid identity with amino acids 874-1110 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 874-1110 of SEQ ID NO: 1. Inother aspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidsubstitutions relative to amino acids 874-1110 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 874-1110 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 874-1110 of SEQ ID NO: 1. In stillother aspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 874-1110 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 874-1110 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 874-1110 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 874-1110 of SEQ ID NO: 1. In yetother aspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 874-1110 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 874-1110 of SEQ ID NO: 1. In stillother aspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 874-1110 of SEQ ID NO: 1. In otheraspects of this embodiment, a BoNT/A H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 874-1110 of SEQ ID NO: 1.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/B H_(CN) region. In an aspect of this embodiment, aBoNT/B H_(CN) region comprises amino acids 861-1097 of SEQ ID NO: 2. Inanother aspect of this embodiment, a BoNT/B H_(CN) region comprises anaturally occurring BoNT/B H_(CN) region variant, such as, e.g., aH_(CN) region from a BoNT/B isoform or a H_(CN) region from a BoNT/Bsubtype. In another aspect of this embodiment, a BoNT/B H_(CN) regioncomprises amino acids 861-1097 of a naturally occurring BoNT/B H_(CN)region variant of SEQ ID NO: 2, such as, e.g., amino acids 861-1097 of aBoNT/B isoform of SEQ ID NO: 2 or amino acids 861-1097 of a BoNT/Bsubtype of SEQ ID NO: 2. In still another aspect of this embodiment, aBoNT/B H_(CN) region comprises a non-naturally occurring BoNT/B H_(CN)region variant, such as, e.g., a conservative BoNT/B H_(CN) regionvariant, a non-conservative BoNT/B H_(CN) region variant, a BoNT/Bchimeric H_(CN) region, an active BoNT/B H_(CN) region fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/B H_(CN) region comprises amino acids 861-1097 of a non-naturallyoccurring BoNT/B H_(CN) region variant of SEQ ID NO: 2, such as, e.g.,amino acids 861-1097 of a conservative BoNT/B H_(CN) region variant ofSEQ ID NO: 2, amino acids 861-1097 of a non-conservative BoNT/B H_(CN)region variant of SEQ ID NO: 2, amino acids 861-1097 of an active BoNT/BH_(CN) region fragment of SEQ ID NO: 2, or any combination thereof.

In other aspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 861-1097 of SEQ ID NO: 2, at least 75% amino acid identity withamino acids 861-1097 of SEQ ID NO: 2, at least 80% amino acid identitywith amino acids 861-1097 of SEQ ID NO: 2, at least 85% amino acididentity with amino acids 861-1097 of SEQ ID NO: 2, at least 90% aminoacid identity with amino acids 861-1097 of SEQ ID NO: 2 or at least 95%amino acid identity with amino acids 861-1097 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 861-1097 of SEQ ID NO: 2, at most 75% amino acid identity withamino acids 861-1097 of SEQ ID NO: 2, at most 80% amino acid identitywith amino acids 861-1097 of SEQ ID NO: 2, at most 85% amino acididentity with amino acids 861-1097 of SEQ ID NO: 2, at most 90% aminoacid identity with amino acids 861-1097 of SEQ ID NO: 2 or at most 95%amino acid identity with amino acids 861-1097 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidsubstitutions relative to amino acids 861-1097 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidsubstitutions relative to amino acids 861-1097 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 861-1097 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 861-1097 of SEQ ID NO: 2. In stillother aspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 861-1097 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 861-1097 of SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 861-1097 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 861-1097 of SEQ ID NO: 2. In yetother aspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 861-1097 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 861-1097 of SEQ ID NO: 2. In stillother aspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 861-1097 of SEQ ID NO: 2. In otheraspects of this embodiment, a BoNT/B H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 861-1097 of SEQ ID NO: 2.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/C1 H_(CN) region. In an aspect of this embodiment, aBoNT/C1 H_(CN) region comprises amino acids 869-1111 of SEQ ID NO: 3. Inanother aspect of this embodiment, a BoNT/C1 H_(CN) region comprises anaturally occurring BoNT/C1 H_(CN) region variant, such as, e.g., aH_(CN) region from a BoNT/C1 isoform or a H_(CN) region from a BoNT/C1subtype. In another aspect of this embodiment, a BoNT/C1 H_(CN) regioncomprises amino acids 869-1111 of a naturally occurring BoNT/C1 H_(CN)region variant of SEQ ID NO: 3, such as, e.g., amino acids 869-1111 of aBoNT/C1 isoform of SEQ ID NO: 3 or amino acids 869-1111 of a BoNT/C1subtype of SEQ ID NO: 3. In still another aspect of this embodiment, aBoNT/C1 H_(CN) region comprises a non-naturally occurring BoNT/C1 H_(CN)region variant, such as, e.g., a conservative BoNT/C1 H_(CN) regionvariant, a non-conservative BoNT/C1 H_(CN) region variant, a BoNT/C1chimeric H_(CN) region, an active BoNT/C1 H_(CN) region fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/C1 H_(CN) region comprises amino acids 869-1111 of a non-naturallyoccurring BoNT/C1 H_(CN) region variant of SEQ ID NO: 3, such as, e.g.,amino acids 869-1111 of a conservative BoNT/C1 H_(CN) region variant ofSEQ ID NO: 3, amino acids 869-1111 of a non-conservative BoNT/C1 H_(CN)region variant of SEQ ID NO: 3, amino acids 869-1111 of an activeBoNT/C1 H_(CN) region fragment of SEQ ID NO: 3, or any combinationthereof.

In other aspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 869-1111 of SEQ ID NO: 3, at least 75% amino acid identity withamino acids 869-1111 of SEQ ID NO: 3, at least 80% amino acid identitywith amino acids 869-1111 of SEQ ID NO: 3, at least 85% amino acididentity with amino acids 869-1111 of SEQ ID NO: 3, at least 90% aminoacid identity with amino acids 869-1111 of SEQ ID NO: 3 or at least 95%amino acid identity with amino acids 869-1111 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 869-1111 of SEQ ID NO: 3, at most 75% amino acid identity withamino acids 869-1111 of SEQ ID NO: 3, at most 80% amino acid identitywith amino acids 869-1111 of SEQ ID NO: 3, at most 85% amino acididentity with amino acids 869-1111 of SEQ ID NO: 3, at most 90% aminoacid identity with amino acids 869-1111 of SEQ ID NO: 3 or at most 95%amino acid identity with amino acids 869-1111 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 869-1111 of SEQ ID NO: 3. Inother aspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidsubstitutions relative to amino acids 869-1111 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 869-1111 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 869-1111 of SEQ ID NO: 3. In stillother aspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 869-1111 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 869-1111 of SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 869-1111 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 869-1111 of SEQ ID NO: 3. In yetother aspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 869-1111 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 869-1111 of SEQ ID NO: 3. In stillother aspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 869-1111 of SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 869-1111 of SEQ ID NO: 3.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/D H_(CN) region. In an aspect of this embodiment, aBoNT/D H_(CN) region comprises amino acids 865-1098 of SEQ ID NO: 4. Inanother aspect of this embodiment, a BoNT/D H_(CN) region comprises anaturally occurring BoNT/D H_(CN) region variant, such as, e.g., aH_(CN) region from a BoNT/D isoform or a H_(CN) region from a BoNT/Dsubtype. In another aspect of this embodiment, a BoNT/D H_(CN) regioncomprises amino acids 865-1098 of a naturally occurring BoNT/D H_(CN)region variant of SEQ ID NO: 4, such as, e.g., amino acids 865-1098 of aBoNT/D isoform of SEQ ID NO: 4 or amino acids 865-1098 of a BoNT/Dsubtype of SEQ ID NO: 4. In still another aspect of this embodiment, aBoNT/D H_(CN) region comprises a non-naturally occurring BoNT/D H_(CN)region variant, such as, e.g., a conservative BoNT/D H_(CN) regionvariant, a non-conservative BoNT/D H_(CN) region variant, a BoNT/Dchimeric H_(CN) region, an active BoNT/D H_(CN) region fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/D H_(CN) region comprises amino acids 865-1098 of a non-naturallyoccurring BoNT/D H_(CN) region variant of SEQ ID NO: 4, such as, e.g.,amino acids 865-1098 of a conservative BoNT/D H_(CN) region variant ofSEQ ID NO: 4, amino acids 865-1098 of a non-conservative BoNT/D H_(CN)region variant of SEQ ID NO: 4, amino acids 865-1098 of an active BoNT/DH_(CN) region fragment of SEQ ID NO: 4, or any combination thereof.

In other aspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 865-1098 of SEQ ID NO: 4, at least 75% amino acid identity withamino acids 865-1098 of SEQ ID NO: 4, at least 80% amino acid identitywith amino acids 865-1098 of SEQ ID NO: 4, at least 85% amino acididentity with amino acids 865-1098 of SEQ ID NO: 4, at least 90% aminoacid identity with amino acids 865-1098 of SEQ ID NO: 4 or at least 95%amino acid identity with amino acids 865-1098 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 865-1098 of SEQ ID NO: 4, at most 75% amino acid identity withamino acids 865-1098 of SEQ ID NO: 4, at most 80% amino acid identitywith amino acids 865-1098 of SEQ ID NO: 4, at most 85% amino acididentity with amino acids 865-1098 of SEQ ID NO: 4, at most 90% aminoacid identity with amino acids 865-1098 of SEQ ID NO: 4 or at most 95%amino acid identity with amino acids 865-1098 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 865-1098 of SEQ ID NO: 4. Inother aspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidsubstitutions relative to amino acids 865-1098 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 865-1098 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 865-1098 of SEQ ID NO: 4. In stillother aspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 865-1098 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 865-1098 of SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 865-1098 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 865-1098 of SEQ ID NO: 4. In yetother aspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 865-1098 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 865-1098 of SEQ ID NO: 4. In stillother aspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 865-1098 of SEQ ID NO: 4. In otheraspects of this embodiment, a BoNT/D H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 865-1098 of SEQ ID NO: 4.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/E H_(CN) region. In an aspect of this embodiment, aBoNT/E H_(CN) region comprises amino acids 848-1085 of SEQ ID NO: 5. Inanother aspect of this embodiment, a BoNT/E H_(CN) region comprises anaturally occurring BoNT/E H_(CN) region variant, such as, e.g., aH_(CN) region from a BoNT/E isoform or a H_(CN) region from a BoNT/Esubtype. In another aspect of this embodiment, a BoNT/E H_(CN) regioncomprises amino acids 848-1085 of a naturally occurring BoNT/E H_(CN)region variant of SEQ ID NO: 5, such as, e.g., amino acids 848-1085 of aBoNT/E isoform of SEQ ID NO: 5 or amino acids 848-1085 of a BoNT/Esubtype of SEQ ID NO: 5. In still another aspect of this embodiment, aBoNT/E H_(CN) region comprises a non-naturally occurring BoNT/E H_(CN)region variant, such as, e.g., a conservative BoNT/E H_(CN) regionvariant, a non-conservative BoNT/E H_(CN) region variant, a BoNT/Echimeric H_(CN) region, an active BoNT/E H_(CN) region fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/E H_(CN) region comprises amino acids 848-1085 of a non-naturallyoccurring BoNT/E H_(CN) region variant of SEQ ID NO: 5, such as, e.g.,amino acids 848-1085 of a conservative BoNT/E H_(CN) region variant ofSEQ ID NO: 5, amino acids 848-1085 of a non-conservative BoNT/E H_(CN)region variant of SEQ ID NO: 5, amino acids 848-1085 of an active BoNT/EH_(CN) region fragment of SEQ ID NO: 5, or any combination thereof.

In other aspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 848-1085 of SEQ ID NO: 5, at least 75% amino acid identity withamino acids 848-1085 of SEQ ID NO: 5, at least 80% amino acid identitywith amino acids 848-1085 of SEQ ID NO: 5, at least 85% amino acididentity with amino acids 848-1085 of SEQ ID NO: 5, at least 90% aminoacid identity with amino acids 848-1085 of SEQ ID NO: 5 or at least 95%amino acid identity with amino acids 848-1085 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 848-1085 of SEQ ID NO: 5, at most 75% amino acid identity withamino acids 848-1085 of SEQ ID NO: 5, at most 80% amino acid identitywith amino acids 848-1085 of SEQ ID NO: 5, at most 85% amino acididentity with amino acids 848-1085 of SEQ ID NO: 5, at most 90% aminoacid identity with amino acids

-   848-1085 of SEQ ID NO: 5 or at most 95% amino acid identity with    amino acids 848-1085 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 848-1085 of SEQ ID NO: 5. Inother aspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidsubstitutions relative to amino acids 848-1085 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 848-1085 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 848-1085 of SEQ ID NO: 5. In stillother aspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 848-1085 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 848-1085 of SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 848-1085 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 848-1085 of SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 848-1085 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 848-1085 of SEQ ID NO: 5. In stillother aspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 848-1085 of SEQ ID NO: 5. In otheraspects of this embodiment, a BoNT/E H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 848-1085 of SEQ ID NO: 5.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/F H_(CN) region. In an aspect of this embodiment, aBoNT/F H_(CN) region comprises amino acids 867-1105 of SEQ ID NO: 6. Inanother aspect of this embodiment, a BoNT/F H_(CN) region comprises anaturally occurring BoNT/F H_(CN) region variant, such as, e.g., aH_(CN) region from a BoNT/F isoform or a H_(CN) region from a BoNT/Fsubtype. In another aspect of this embodiment, a BoNT/F H_(CN) regioncomprises amino acids 867-1105 of a naturally occurring BoNT/F H_(CN)region variant of SEQ ID NO: 6, such as, e.g., amino acids 867-1105 of aBoNT/F isoform of SEQ ID NO: 6 or amino acids 867-1105 of a BoNT/Fsubtype of SEQ ID NO: 6. In still another aspect of this embodiment, aBoNT/F H_(CN) region comprises a non-naturally occurring BoNT/F H_(CN)region variant, such as, e.g., a conservative BoNT/F H_(CN) regionvariant, a non-conservative BoNT/F H_(CN) region variant, a BoNT/Fchimeric H_(CN) region, an active BoNT/F H_(CN) region fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/F H_(CN) region comprises amino acids 867-1105 of a non-naturallyoccurring BoNT/F H_(CN) region variant of SEQ ID NO: 6, such as, e.g.,amino acids 867-1105 of a conservative BoNT/F H_(CN) region variant ofSEQ ID NO: 6, amino acids 867-1105 of a non-conservative BoNT/F H_(CN)region variant of SEQ ID NO: 6, amino acids 867-1105 of an active BoNT/FH_(CN) region fragment of SEQ ID NO: 6, or any combination thereof.

In other aspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 867-1105 of SEQ ID NO: 6, at least 75% amino acid identity withamino acids 867-1105 of SEQ ID NO: 6, at least 80% amino acid identitywith amino acids 867-1105 of SEQ ID NO: 6, at least 85% amino acididentity with amino acids 867-1105 of SEQ ID NO: 6, at least 90% aminoacid identity with amino acids 867-1105 of SEQ ID NO: 6 or at least 95%amino acid identity with amino acids 867-1105 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 867-1105 of SEQ ID NO: 6, at most 75% amino acid identity withamino acids 867-1105 of SEQ ID NO: 6, at most 80% amino acid identitywith amino acids 867-1105 of SEQ ID NO: 6, at most 85% amino acididentity with amino acids 867-1105 of SEQ ID NO: 6, at most 90% aminoacid identity with amino acids 867-1105 of SEQ ID NO: 6 or at most 95%amino acid identity with amino acids 867-1105 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 867-1105 of SEQ ID NO: 6. Inother aspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidsubstitutions relative to amino acids 867-1105 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 867-1105 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 867-1105 of SEQ ID NO: 6. In stillother aspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 867-1105 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 867-1105 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 867-1105 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 867-1105 of SEQ ID NO: 6. In yetother aspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 867-1105 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 867-1105 of SEQ ID NO: 6. In stillother aspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 867-1105 of SEQ ID NO: 6. In otheraspects of this embodiment, a BoNT/F H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 867-1105 of SEQ ID NO: 6.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/G H_(CN) region. In an aspect of this embodiment, aBoNT/G H_(CN) region comprises amino acids 866-1105 of SEQ ID NO: 7. Inanother aspect of this embodiment, a BoNT/G H_(CN) region comprises anaturally occurring BoNT/G H_(CN) region variant, such as, e.g., aH_(CN) region from a BoNT/G isoform or a H_(CN) region from a BoNT/Gsubtype. In another aspect of this embodiment, a BoNT/G H_(CN) regioncomprises amino acids 866-1105 of a naturally occurring BoNT/G H_(CN)region variant of SEQ ID NO: 7, such as, e.g., amino acids 866-1105 of aBoNT/G isoform of SEQ ID NO: 7 or amino acids 866-1105 of a BoNT/Gsubtype of SEQ ID NO: 7. In still another aspect of this embodiment, aBoNT/G H_(CN) region comprises a non-naturally occurring BoNT/G H_(CN)region variant, such as, e.g., a conservative BoNT/G H_(CN) regionvariant, a non-conservative BoNT/G H_(CN) region variant, a BoNT/Gchimeric H_(CN) region, an active BoNT/G H_(CN) region fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/G H_(CN) region comprises amino acids 866-1105 of a non-naturallyoccurring BoNT/G H_(CN) region variant of SEQ ID NO: 7, such as, e.g.,amino acids 866-1105 of a conservative BoNT/G H_(CN) region variant ofSEQ ID NO: 7, amino acids 866-1105 of a non-conservative BoNT/G H_(CN)region variant of SEQ ID NO: 7, amino acids 866-1105 of an active BoNT/GH_(CN) region fragment of SEQ ID NO: 7, or any combination thereof.

In other aspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 866-1105 of SEQ ID NO: 7, at least 75% amino acid identity withamino acids 866-1105 of SEQ ID NO: 7, at least 80% amino acid identitywith amino acids 866-1105 of SEQ ID NO: 7, at least 85% amino acididentity with amino acids 866-1105 of SEQ ID NO: 7, at least 90% aminoacid identity with amino acids 866-1105 of SEQ ID NO: 7 or at least 95%amino acid identity with amino acids 866-1105 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 866-1105 of SEQ ID NO: 7, at most 75% amino acid identity withamino acids 866-1105 of SEQ ID NO: 7, at most 80% amino acid identitywith amino acids 866-1105 of SEQ ID NO: 7, at most 85% amino acididentity with amino acids 866-1105 of SEQ ID NO: 7, at most 90% aminoacid identity with amino acids 866-1105 of SEQ ID NO: 7 or at most 95%amino acid identity with amino acids 866-1105 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 866-1105 of SEQ ID NO: 7. Inother aspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidsubstitutions relative to amino acids 866-1105 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 866-1105 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 866-1105 of SEQ ID NO: 7. In stillother aspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 866-1105 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidadditions relative to amino acids 866-1105 of SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 866-1105 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 866-1105 of SEQ ID NO: 7. In yetother aspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 866-1105 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino aciddeletions relative to amino acids 866-1105 of SEQ ID NO: 7. In stillother aspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 866-1105 of SEQ ID NO: 7. In otheraspects of this embodiment, a BoNT/G H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidadditions relative to amino acids 866-1105 of SEQ ID NO: 7.

In another embodiment, a Clostridial toxin translocation domaincomprises a TeNT H_(CN) region. In an aspect of this embodiment, a TeNTH_(CN) region comprises amino acids 882-1127 of SEQ ID NO: 8. In anotheraspect of this embodiment, a TeNT H_(CN) region comprises a naturallyoccurring TeNT H_(CN) region variant, such as, e.g., a H_(CN) regionfrom a TeNT isoform or a H_(CN) region from a TeNT subtype. In anotheraspect of this embodiment, a TeNT H_(CN) region comprises amino acids882-1127 of a naturally occurring TeNT H_(CN) region variant of SEQ IDNO: 8, such as, e.g., amino acids 882-1127 of a TeNT isoform of SEQ IDNO: 8 or amino acids 882-1127 of a TeNT subtype of SEQ ID NO: 8. Instill another aspect of this embodiment, a TeNT H_(CN) region comprisesa non-naturally occurring TeNT H_(CN) region variant, such as, e.g., aconservative TeNT H_(CN) region variant, a non-conservative TeNT H_(CN)region variant, a TeNT chimeric H_(CN) region, an active TeNT H_(CN)region fragment, or any combination thereof. In still another aspect ofthis embodiment, a TeNT H_(CN) region comprises amino acids 882-1127 ofa non-naturally occurring TeNT H_(CN) region variant of SEQ ID NO: 8,such as, e.g., amino acids 882-1127 of a conservative TeNT H_(CN) regionvariant of SEQ ID NO: 8, amino acids 882-1127 of a non-conservative TeNTH_(CN) region variant of SEQ ID NO: 8, amino acids 882-1127 of an activeTeNT H_(CN) region fragment of SEQ ID NO: 8, or any combination thereof.

In other aspects of this embodiment, a TeNT H_(CN) region comprises apolypeptide having, e.g., at least 70% amino acid identity with aminoacids 882-1127 of SEQ ID NO: 8, at least 75% amino acid identity withamino acids 882-1127 of SEQ ID NO: 8, at least 80% amino acid identitywith amino acids 882-1127 of SEQ ID NO: 8, at least 85% amino acididentity with amino acids 882-1127 of SEQ ID NO: 8, at least 90% aminoacid identity with amino acids 882-1127 of SEQ ID NO: 8 or at least 95%amino acid identity with amino acids 882-1127 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT H_(CN) region comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 882-1127 of SEQ ID NO: 8, at most 75% amino acid identity withamino acids 882-1127 of SEQ ID NO: 8, at most 80% amino acid identitywith amino acids 882-1127 of SEQ ID NO: 8, at most 85% amino acididentity with amino acids 882-1127 of SEQ ID NO: 8, at most 90% aminoacid identity with amino acids 882-1127 of SEQ ID NO: 8 or at most 95%amino acid identity with amino acids 882-1127 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, or 200 non-contiguous aminoacid substitutions relative to amino acids 882-1127 of SEQ ID NO: 8. Inother aspects of this embodiment, a TeNT H_(CN) region comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acidsubstitutions relative to amino acids 882-1127 of SEQ ID NO: 8. In yetother aspects of this embodiment, a TeNT H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino aciddeletions relative to amino acids 882-1127 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT H_(CN) region comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50 or 100 non-contiguous amino acid deletionsrelative to amino acids 882-1127 of SEQ ID NO: 8. In still other aspectsof this embodiment, a TeNT H_(CN) region comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50 or 100 non-contiguous amino acid additions relative toamino acids 882-1127 of SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT H_(CN) region comprises a polypeptide having, e.g.,at least one, two, three, four, five, six, seven, eight, nine, 10, 20,30, 40, 50 or 100 non-contiguous amino acid additions relative to aminoacids 882-1127 of SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT H_(CN) region comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguous amino acidsubstitutions relative to amino acids 882-1127 of SEQ ID NO: 8. In otheraspects of this embodiment, a TeNT H_(CN) region comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50 or 100 contiguous amino acid substitutionsrelative to amino acids 882-1127 of SEQ ID NO: 8. In yet other aspectsof this embodiment, a TeNT H_(CN) region comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50 or 100 contiguous amino acid deletions relative to aminoacids 882-1127 of SEQ ID NO: 8. In other aspects of this embodiment, aTeNT H_(CN) region comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50 or100 contiguous amino acid deletions relative to amino acids 882-1127 ofSEQ ID NO: 8. In still other aspects of this embodiment, a TeNT H_(CN)region comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50 or 100contiguous amino acid additions relative to amino acids 882-1127 of SEQID NO: 8. In other aspects of this embodiment, a TeNT H_(CN) regioncomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50 or 100 contiguousamino acid additions relative to amino acids 882-1127 of SEQ ID NO: 8.

The fusion of the membrane of enveloped viruses to a cellular membraneis an essential step in the release of the viral capsule into thecytoplasm of the host cell. This fusion event is mediated by a fusogenicpeptide segment present in viral glycoproteins located on the viralmembrane and involves either a pH-dependent or pH-independent process,see, e.g., Frederick M. Hughson, Structural Characterization of ViralFusion Proteins, 5(3) Curr. Biol. 158-274 (1128); Trudy G. Morrison,Structure and Function of a Paramyxovirus Fusion Protein, 1304(1)Biochim. Biophys. Acta. 73-84; David J. Schibli and WinfriedWeissenhorn, Class I and Class II Viral Fusion Protein Structures RevealSimilar Principles in Membrane Fusion, 21(6) Mol. Membr. Biol. 361-371(1137). The fusogenic peptide domain comprises a hydrophobic,glycine-rich peptide of approximately 20-30 amino acids that assist inthe insertion of the viral capsule into a cellular membrane. Thus, anenveloped virus fusogenic peptide domain can be useful as atranslocation facilitating domain.

An enveloped virus fusogenic peptide domain includes, withoutlimitation, naturally occurring enveloped virus fusogenic peptide domainvariants, such as, e.g., enveloped virus fusogenic peptide domainisoforms and enveloped virus fusogenic peptide domain subtypes;non-naturally occurring enveloped virus fusogenic peptide domainvariants, such as, e.g., conservative enveloped virus fusogenic peptidedomain variants, non-conservative enveloped virus fusogenic peptidedomain variants, enveloped virus fusogenic peptide domain chimerics,active enveloped virus fusogenic peptide domain fragments thereof, orany combination thereof.

As used herein, the term “enveloped virus fusogenic peptide domainvariant,” whether naturally-occurring or non-naturally-occurring, meansan enveloped virus fusogenic peptide domain that has at least one aminoacid change from the corresponding region of the disclosed referencesequences and can be described in percent identity to the correspondingregion of that reference sequence. Unless expressly indicated, allClostridial toxin H_(CN) region variants disclosed in the presentspecification are capable of further facilitating the translocation stepof the intoxication process that mediates Clostridial toxin light chaintranslocation. As non-limiting examples, an Influenza virus A fusogenicpeptide domain variant comprising SEQ ID NO: 87 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to SEQ ID NO: 87; a Semliki Forestvirus fusogenic peptide domain variant comprising SEQ ID NO: 92 willhave at least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to SEQ ID NO: 92; anEastern equine encephalitis virus fusogenic peptide domain variantcomprising SEQ ID NO: 94 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to SEQ ID NO: 94; a Venezuelan equine encephalitis virusfusogenic peptide domain variant comprising SEQ ID NO: 102 will have atleast one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to SEQ ID NO: 102; aVesicular stomatitis virus fusogenic peptide domain variant comprisingSEQ ID NO: 119 will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared toSEQ ID NO: 119; a Sendai virus fusogenic peptide domain variantcomprising SEQ ID NO: 130 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to SEQ ID NO: 130; a Canine distemper virus fusogenic peptidedomain variant comprising SEQ ID NO: 137 will have at least one aminoacid difference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to SEQ ID NO: 137; a Newcastle disease virusfusogenic peptide domain variant comprising SEQ ID NO: 147 will have atleast one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to SEQ ID NO: 147; and aHendra virus fusogenic peptide domain variant comprising SEQ ID NO: 160will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to SEQ ID NO: 160.

It is recognized by those of skill in the art that for each envelopedvirus there can be naturally occurring fusogenic peptide domain variantsthat differ somewhat in their amino acid sequence, and also in thenucleic acids encoding these proteins. For example, at least fivenaturally-occurring variants of the fusogenic peptide domain present inthe Influenza A virus haemagglutinin are known (SEQ ID NO: 87 to SEQ IDNO: 91); at least six naturally-occurring variants of the fusogenicpeptide domain present in the Eastern equine encephalitis virus E1protein are known (SEQ ID NO: 94 to SEQ ID NO: 99); at least eightnaturally-occurring variants of the fusogenic peptide domain present inthe Venezuelan equine encephalitis virus E1 protein are known (SEQ IDNO: 102 to SEQ ID NO: 109); at least seven naturally-occurring variantsof the fusogenic peptide domain present in the Vesicular stomatitisvirus (VSV) glycoprotein G are known (SEQ ID NO: 119 to SEQ ID NO: 125);and at least eleven naturally-occurring variants of the fusogenicpeptide domain present in the Newcastle disease virus F protein areknown (SEQ ID NO: 147 to SEQ ID NO: 157). As used herein, the term“enveloped virus fusogenic peptide domain variant” means any envelopedvirus fusogenic peptide domain produced by a naturally-occurringprocess, including, without limitation, enveloped virus fusogenicpeptide domain isoforms produced from alternatively-spliced transcripts,enveloped virus fusogenic peptide domain isoforms produced byspontaneous mutation and enveloped virus fusogenic peptide domainsubtypes. A naturally occurring enveloped virus fusogenic peptide domainvariant can function in substantially the same manner as the referenceenveloped virus fusogenic peptide domain on which the naturallyoccurring enveloped virus fusogenic peptide domain variant is based, andcan be substituted for the reference enveloped virus fusogenic peptidedomain in any aspect of the present invention. A naturally occurringenveloped virus fusogenic peptide domain variant may substitute one ormore amino acids, two or more amino acids, three or more amino acids,four or more amino acids, five or more amino acids or ten or more aminoacids from the reference enveloped virus fusogenic peptide domain onwhich the naturally occurring enveloped virus fusogenic peptide domainis based. A naturally occurring enveloped virus fusogenic peptide domainvariant can also substitute at least 2 contiguous amino acids, at least3 contiguous amino acids, at least 4 contiguous amino acids or at least5 contiguous amino acids from the reference enveloped virus fusogenicpeptide domain on which the naturally occurring enveloped virusfusogenic peptide domain variant is based, that possess at least 50%amino acid identity, 65% amino acid identity, 75% amino acid identity,85% amino acid identity or 95% amino acid identity to the referenceenveloped virus fusogenic peptide domain on which the naturallyoccurring enveloped virus fusogenic peptide domain variant is based.

A non-limiting examples of a naturally occurring enveloped virusfusogenic peptide domain variant is a enveloped virus fusogenic peptidedomain isoform such as, e.g., an influenzavirus fusogenic peptide domainisoform, an alphavirus fusogenic peptide domain isoform, a vesiculovirusfusogenic peptide domain isoform, a respirovirus fusogenic peptidedomain isoform, a morbillivirus fusogenic peptide domain isoform, anavulavirus fusogenic peptide domain isoform, a henipavirus fusogenicpeptide domain isoform, a metapneumovirus fusogenic peptide domainisoform and a foamy virus fusogenic peptide domain isoform. An envelopedvirus fusogenic peptide domain isoform can function in substantially thesame manner as the reference enveloped virus fusogenic peptide domain onwhich the enveloped virus fusogenic peptide domain isoform is based, andcan be substituted for the reference enveloped virus fusogenic peptidedomain in any aspect of the present invention.

A non-limiting examples of a naturally occurring enveloped virusfusogenic peptide domain variant is a enveloped virus fusogenic peptidedomain subtype such as, e.g., an influenzavirus fusogenic peptide domainsubtype, an alphavirus fusogenic peptide domain subtype, a vesiculovirusfusogenic peptide domain subtype, a respirovirus fusogenic peptidedomain subtype, a morbillivirus fusogenic peptide domain subtype, anavulavirus fusogenic peptide domain subtype, a henipavirus fusogenicpeptide domain subtype, a metapneumovirus fusogenic peptide domainsubtype and a foamy virus fusogenic peptide domain subtype. An envelopedvirus fusogenic peptide domain subtype can function in substantially thesame manner as the reference enveloped virus fusogenic peptide domain onwhich the enveloped virus fusogenic peptide domain subtype is based, andcan be substituted for the reference enveloped virus fusogenic peptidedomain in any aspect of the present invention.

As used herein, the term “non-naturally occurring enveloped virusfusogenic peptide domain variant” means any enveloped virus fusogenicpeptide domain produced with the aid of human manipulation, including,without limitation, enveloped virus fusogenic peptide domains producedby genetic engineering using random mutagenesis or rational design andenveloped virus fusogenic peptide domains produced by chemicalsynthesis. Non-limiting examples of non-naturally occurring envelopedvirus fusogenic peptide domain variants include, e.g., conservativeenveloped virus fusogenic peptide domain variants, non-conservativeenveloped virus fusogenic peptide domain variants, enveloped virusfusogenic peptide domain chimeric variants and active enveloped virusfusogenic peptide domain fragments.

As used herein, the term “conservative enveloped virus fusogenic peptidedomain variant” means a enveloped virus fusogenic peptide domain thathas at least one amino acid substituted by another amino acid or anamino acid analog that has at least one property similar to that of theoriginal amino acid from the reference enveloped virus fusogenic peptidedomain sequence. Examples of properties include, without limitation,similar size, topography, charge, hydrophobicity, hydrophilicity,lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, aphysicochemical property, of the like, or any combination thereof. Aconservative enveloped virus fusogenic peptide domain variant canfunction in substantially the same manner as the reference envelopedvirus fusogenic peptide domain on which the conservative enveloped virusfusogenic peptide domain variant is based, and can be substituted forthe reference enveloped virus fusogenic peptide domain in any aspect ofthe present invention. A conservative enveloped virus fusogenic peptidedomain variant may substitute one or more amino acids, two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids or ten or more amino acids from the reference envelopedvirus fusogenic peptide domain on which the conservative enveloped virusfusogenic peptide domain variant is based. A conservative envelopedvirus fusogenic peptide domain variant can also substitute at least 2contiguous amino acids, at least 3 contiguous amino acids, at least 4contiguous amino acids or at least 5 contiguous amino acids from thereference enveloped virus fusogenic peptide domain on which theconservative enveloped virus fusogenic peptide domain variant is based,that possess at least 50% amino acid identity, 65% amino acid identity,75% amino acid identity, 85% amino acid identity or 95% amino acididentity to the reference enveloped virus fusogenic peptide domain onwhich the conservative enveloped virus fusogenic peptide domain variantis based. Non-limiting examples of a conservative enveloped virusfusogenic peptide domain variant include, e.g., conservativeinfluenzavirus fusogenic peptide domain variants, conservativealphavirus fusogenic peptide domain variants, conservative vesiculovirusfusogenic peptide domain variants, conservative respirovirus fusogenicpeptide domain variants, conservative morbillivirus fusogenic peptidedomain variants, conservative avulavirus fusogenic peptide domainvariants, conservative henipavirus fusogenic peptide domain variants,conservative metapneumovirus fusogenic peptide domain variants andconservative foamy virus fusogenic peptide domain variants.

As used herein, the term “non-conservative enveloped virus fusogenicpeptide domain variant” means a enveloped virus fusogenic peptide domainin which 1) at least one amino acid is deleted from the referenceenveloped virus fusogenic peptide domain on which the non-conservativeenveloped virus fusogenic peptide domain variant is based; 2) at leastone amino acid added to the reference enveloped virus fusogenic peptidedomain on which the non-conservative enveloped virus fusogenic peptidedomain is based; or 3) at least one amino acid is substituted by anotheramino acid or an amino acid analog that does not share any propertysimilar to that of the original amino acid from the reference envelopedvirus fusogenic peptide domain sequence. A non-conservative envelopedvirus fusogenic peptide domain variant can function in substantially thesame manner as the reference enveloped virus fusogenic peptide domain onwhich the non-conservative enveloped virus fusogenic peptide domainvariant is based, and can be substituted for the reference envelopedvirus fusogenic peptide domain in any aspect of the present invention. Anon-conservative enveloped virus fusogenic peptide domain variant candelete one or more amino acids, two or more amino acids, three or moreamino acids, four or more amino acids or five or more amino acids fromthe reference enveloped virus fusogenic peptide domain on which thenon-conservative enveloped virus fusogenic peptide domain variant isbased. A non-conservative enveloped virus fusogenic peptide domainvariant can add one or more amino acids, two or more amino acids, threeor more amino acids, four or more amino acids or five or more aminoacids to the reference enveloped virus fusogenic peptide domain on whichthe non-conservative enveloped virus fusogenic peptide domain variant isbased. A non-conservative enveloped virus fusogenic peptide domainvariant may substitute one or more amino acids, two or more amino acids,three or more amino acids, four or more amino acids, five or more aminoacids or ten or more amino acids from the reference enveloped virusfusogenic peptide domain on which the non-conservative enveloped virusfusogenic peptide domain variant is based. A non-conservative envelopedvirus fusogenic peptide domain variant can also substitute at least 2contiguous amino acids, at least 3 contiguous amino acids, at least 4contiguous amino acids or at least 5 contiguous amino acids from thereference enveloped virus fusogenic peptide domain on which thenon-conservative enveloped virus fusogenic peptide domain variant isbased, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference enveloped virus fusogenic peptide domainon which the non-conservative enveloped virus fusogenic peptide domainvariant is based. Non-limiting examples of a non-conservative envelopedvirus fusogenic peptide domain variant include, e.g., non-conservativeinfluenzavirus fusogenic peptide domain variants, non-conservativealphavirus fusogenic peptide domain variants, non-conservativevesiculovirus fusogenic peptide domain variants, non-conservativerespirovirus fusogenic peptide domain variants, non-conservativemorbillivirus fusogenic peptide domain variants, non-conservativeavulavirus fusogenic peptide domain variants, non-conservativehenipavirus fusogenic peptide domain variants, non-conservativemetapneumovirus fusogenic peptide domain variants and non-conservativefoamy virus fusogenic peptide domain variants.

As used herein, the term “enveloped virus fusogenic peptide domainchimeric” means a polypeptide comprising at least a portion of anenveloped virus fusogenic peptide domain and at least a portion of atleast one other polypeptide to form an enveloped virus fusogenic peptidedomain with at least one property different from the reference envelopedvirus fusogenic peptide domain, with the proviso that this envelopedvirus fusogenic peptide domain chimeric is still capable of furtherfacilitating the translocation step of the intoxication process wherethe LC is released from intracellular vesicles into the cytoplasm of thetarget cell and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate.

As used herein, the term “active enveloped virus fusogenic peptidedomain fragment” means any of a variety of enveloped virus fusogenicpeptide domain fragments that can further facilitate the translocationstep of the intoxication process where the LC is released fromintracellular vesicles into the cytoplasm of the target cell and thusparticipate in executing the overall cellular mechanism whereby aClostridial toxin proteolytically cleaves a substrate. Enveloped virusfusogenic peptide domains are approximately 15-30 amino acids in length.Thus, aspects of this embodiment can include a translocationfacilitating domain comprising an active enveloped virus fusogenicpeptide domain fragment having a length of, e.g., at least 10 aminoacids, at least 15 amino acids, at least 20 amino acids and at least 25amino acids. Other aspects of this embodiment can include atranslocation facilitating domain comprising an active enveloped virusfusogenic peptide domain fragment having a length of, e.g., at most 10amino acids, at most 15 amino acids, at most 20 amino acids and at most25 amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring enveloped virus fusogenicpeptide domain variants and non-naturally-occurring enveloped virusfusogenic peptide domain variants, including, without limitation, globalmethods, local methods and hybrid methods, such as, e.g., segmentapproach methods. Protocols to determine percent identity are routineprocedures within the scope of one skilled in the art and from theteaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin translocationfacilitating domain comprising an enveloped virus fusogenic peptidedomain. In an aspect of this embodiment, a Clostridial toxintranslocation facilitating domain comprises a naturally occurringenveloped virus fusogenic peptide domain variant, such as, e.g., aenveloped virus fusogenic peptide domain isoform or a enveloped virusfusogenic peptide domain subtype. In another aspect of this embodiment,a Clostridial toxin translocation domain comprises a non-naturallyoccurring enveloped virus fusogenic peptide domain variant, such as,e.g., a conservative enveloped virus fusogenic peptide domain variant, anon-conservative enveloped virus fusogenic peptide domain variant, aenveloped virus fusogenic peptide domain chimeric, an active envelopedvirus fusogenic peptide domain fragment, or any combination thereof.

In another embodiment, a Clostridial toxin translocation facilitatingdomain comprises an influenzavirus fusogenic peptide domain. In anotheraspect of this embodiment, an influenzavirus fusogenic peptide domaincomprises a naturally occurring influenzavirus fusogenic peptide domainvariant, such as, e.g., an influenzavirus fusogenic peptide domainisoform or an influenzavirus fusogenic peptide domain subtype. Inanother aspect of this embodiment, an influenzavirus fusogenic peptidedomain comprises a naturally occurring influenzavirus fusogenic peptidedomain variant of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ IDNO: 90 or SEQ ID NO: 91, such as, e.g., an influenzavirus fusogenicpeptide domain isoform of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89,SEQ ID NO: 90 or SEQ ID NO: 91 or an influenzavirus fusogenic peptidedomain subtype of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ IDNO: 90 or SEQ ID NO: 91. In still another aspect of this embodiment, aninfluenzavirus fusogenic peptide domain comprises a non-naturallyoccurring influenzavirus fusogenic peptide domain variant, such as,e.g., a conservative influenzavirus fusogenic peptide domain variant, anon-conservative influenzavirus fusogenic peptide domain variant, aninfluenzavirus fusogenic peptide domain chimeric, an activeinfluenzavirus fusogenic peptide domain fragment, or any combinationthereof. In still another aspect of this embodiment, an influenzavirusfusogenic peptide domain comprises amino acids a non-naturally occurringinfluenzavirus fusogenic peptide domain variant of SEQ ID NO: 87, SEQ IDNO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91, such as, e.g., aconservative influenzavirus fusogenic peptide domain variant of SEQ IDNO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91, anon-conservative influenzavirus fusogenic peptide domain variant of SEQID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91,an active influenzavirus fusogenic peptide domain fragment of SEQ ID NO:87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91, or anycombination thereof.

In other aspects of this embodiment, an influenzavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90or SEQ ID NO: 91, at least 75% amino acid identity with SEQ ID NO: 87,SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91, at least80% amino acid identity with SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO:89, SEQ ID NO: 90 or SEQ ID NO: 91, at least 85% amino acid identitywith SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQID NO: 91, at least 90% amino acid identity with SEQ ID NO: 87, SEQ IDNO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91 or at least 95%amino acid identity with SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89,SEQ ID NO: 90 or SEQ ID NO: 91. In yet other aspects of this embodiment,an influenzavirus fusogenic peptide domain comprises a polypeptidehaving, e.g., at most 70% amino acid identity with SEQ ID NO: 87, SEQ IDNO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91, at most 75% aminoacid identity with SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ IDNO: 90 or SEQ ID NO: 91, at most 80% amino acid identity with SEQ ID NO:87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91, atmost 85% amino acid identity with SEQ ID NO: 87, SEQ ID NO: 88, SEQ IDNO: 89, SEQ ID NO: 90 or SEQ ID NO: 91, at most 90% amino acid identitywith SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQID NO: 91 or at most 95% amino acid identity with SEQ ID NO: 87, SEQ IDNO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91.

In other aspects of this embodiment, an influenzavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89,SEQ ID NO: 90 or SEQ ID NO: 91. In other aspects of this embodiment, aninfluenzavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 non-contiguous amino acid substitutions relative to SEQ ID NO: 87,SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91. In yetother aspects of this embodiment, an influenzavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino aciddeletions relative to SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQID NO: 90 or SEQ ID NO: 91. In other aspects of this embodiment, aninfluenzavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 non-contiguous amino acid deletions relative to SEQ ID NO: 87, SEQ IDNO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91. In still otheraspects of this embodiment, an influenzavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90or SEQ ID NO: 91. In other aspects of this embodiment, an influenzavirusfusogenic peptide domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid additions relative to SEQ ID NO: 87, SEQ IDNO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91.

In other aspects of this embodiment, an influenzavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89,SEQ ID NO: 90 or SEQ ID NO: 91. In other aspects of this embodiment, aninfluenzavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid substitutions relative to SEQ ID NO: 87, SEQ IDNO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91. In yet otheraspects of this embodiment, an influenzavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid deletionsrelative to SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90or SEQ ID NO: 91. In other aspects of this embodiment, an influenzavirusfusogenic peptide domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid deletions relative to SEQ ID NO: 87, SEQ ID NO: 88, SEQ IDNO: 89, SEQ ID NO: 90 or SEQ ID NO: 91. In still other aspects of thisembodiment, an influenzavirus fusogenic peptide domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative to SEQID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91.In other aspects of this embodiment, an influenzavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidadditions relative to SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQID NO: 90 or SEQ ID NO: 91.

In another embodiment, a Clostridial toxin translocation facilitatingdomain comprises an alphavirus fusogenic peptide domain. In anotheraspect of this embodiment, an alphavirus fusogenic peptide domaincomprises a naturally occurring alphavirus fusogenic peptide domainvariant, such as, e.g., an alphavirus fusogenic peptide domain isoformor an alphavirus fusogenic peptide domain subtype. In another aspect ofthis embodiment, an alphavirus fusogenic peptide domain comprises anaturally occurring alphavirus fusogenic peptide domain variant of SEQID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96,SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO:101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118, such as,e.g., an alphavirus fusogenic peptide domain isoform of SEQ ID NO: 92,SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ IDNO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115,SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118 or an alphavirusfusogenic peptide domain subtype of SEQ ID NO: 92, SEQ ID NO: 93, SEQ IDNO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQID NO: 117 or SEQ ID NO: 118. In still another aspect of thisembodiment, an alphavirus fusogenic peptide domain comprises anon-naturally occurring alphavirus fusogenic peptide domain variant,such as, e.g., a conservative alphavirus fusogenic peptide domainvariant, a non-conservative alphavirus fusogenic peptide domain variant,an alphavirus fusogenic peptide domain chimeric, an active alphavirusfusogenic peptide domain fragment, or any combination thereof. In stillanother aspect of this embodiment, an alphavirus fusogenic peptidedomain comprises amino acids a non-naturally occurring alphavirusfusogenic peptide domain variant of SEQ ID NO: 92, SEQ ID NO: 93, SEQ IDNO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQID NO: 117 or SEQ ID NO: 118, such as, e.g., a conservative alphavirusfusogenic peptide domain variant of SEQ ID NO: 92, SEQ ID NO: 93, SEQ IDNO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQID NO: 117 or SEQ ID NO: 118, a non-conservative alphavirus fusogenicpeptide domain variant of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117 or SEQ ID NO: 118, an active alphavirus fusogenic peptide domainfragment of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO:118, or any combination thereof.

In other aspects of this embodiment, an alphavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO:95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ IDNO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104,SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ IDNO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113,SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ IDNO: 118, at least 75% amino acid identity with SEQ ID NO: 92, SEQ ID NO:93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ IDNO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ IDNO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111,SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ IDNO: 116, SEQ ID NO: 117 or SEQ ID NO: 118, at least 80% amino acididentity with SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90or SEQ ID NO: 91, at least 85% amino acid identity with SEQ ID NO: 92,SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ IDNO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115,SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118, at least 90% aminoacid identity with SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ IDNO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO:113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 orSEQ ID NO: 118 or at least 95% amino acid identity with SEQ ID NO: 92,SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ IDNO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115,SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118. In yet other aspectsof this embodiment, an alphavirus fusogenic peptide domain comprises apolypeptide having, e.g., at most 70% amino acid identity with SEQ IDNO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101,SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ IDNO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ IDNO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118, at most 75%amino acid identity with SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117 or SEQ ID NO: 118, at most 80% amino acid identity with SEQ ID NO:92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ IDNO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101,SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ IDNO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ IDNO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118, at most 85%amino acid identity with SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117 or SEQ ID NO: 118, at most 90% amino acid identity with SEQ ID NO:92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ IDNO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101,SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ IDNO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ IDNO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118 or at most 95%amino acid identity with SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117 or SEQ ID NO: 118.

In other aspects of this embodiment, an alphavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117 or SEQ ID NO: 118. In other aspects of this embodiment, analphavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 non-contiguous amino acid substitutions relative to SEQ ID NO: 92,SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ IDNO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115,SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118. In yet other aspectsof this embodiment, an alphavirus fusogenic peptide domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toSEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO:96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ IDNO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105,SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ IDNO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114,SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118. Inother aspects of this embodiment, an alphavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid deletionsrelative to SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO:118. In still other aspects of this embodiment, an alphavirus fusogenicpeptide domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid additions relative to SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117 or SEQ ID NO: 118. In other aspects of this embodiment, analphavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 non-contiguous amino acid additions relative to SEQ ID NO: 92, SEQ IDNO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118.

In other aspects of this embodiment, an alphavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:117 or SEQ ID NO: 118. In other aspects of this embodiment, analphavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid substitutions relative to SEQ ID NO: 92, SEQ IDNO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118. In yet other aspects ofthis embodiment, an alphavirus fusogenic peptide domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid deletions relative to SEQID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96,SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO:101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118. In otheraspects of this embodiment, an alphavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid deletionsrelative to SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO:118. In still other aspects of this embodiment, an alphavirus fusogenicpeptide domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or 10 contiguous amino acidadditions relative to SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99,SEQ ID NO: 100, SEQ ID NO:

-   101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105,    SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ    ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID    NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID    NO: 118. In other aspects of this embodiment, an alphavirus    fusogenic peptide domain comprises a polypeptide having, e.g., at    least one, two, three, four, five, six, seven, eight, nine or 10    contiguous amino acid additions relative to SEQ ID NO: 92, SEQ ID    NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97,    SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID    NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:    106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,    SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ    ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118.

In another embodiment, a Clostridial toxin translocation facilitatingdomain comprises a vesiculovirus fusogenic peptide domain. In anotheraspect of this embodiment, a vesiculovirus fusogenic peptide domaincomprises a naturally occurring vesiculovirus fusogenic peptide domainvariant, such as, e.g., a vesiculovirus fusogenic peptide domain isoformor a vesiculovirus fusogenic peptide domain subtype. In another aspectof this embodiment, a vesiculovirus fusogenic peptide domain comprises anaturally occurring vesiculovirus fusogenic peptide domain variant ofSEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ IDNO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127,SEQ ID NO: 128 or SEQ ID NO: 129, such as, e.g., a vesiculovirusfusogenic peptide domain isoform of SEQ ID NO: 119, SEQ ID NO: 120, SEQID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO:125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129 ora vesiculovirus fusogenic peptide domain subtype of SEQ ID NO: 119, SEQID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 orSEQ ID NO: 129. In still another aspect of this embodiment, avesiculovirus fusogenic peptide domain comprises a non-naturallyoccurring vesiculovirus fusogenic peptide domain variant, such as, e.g.,a conservative vesiculovirus fusogenic peptide domain variant, anon-conservative vesiculovirus fusogenic peptide domain variant, avesiculovirus fusogenic peptide domain chimeric, an active vesiculovirusfusogenic peptide domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a vesiculovirus fusogenic peptidedomain comprises amino acids a non-naturally occurring vesiculovirusfusogenic peptide domain variant of SEQ ID NO: 119, SEQ ID NO: 120, SEQID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO:125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129,such as, e.g., a conservative vesiculovirus fusogenic peptide domainvariant of SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129, a non-conservativevesiculovirus fusogenic peptide domain variant of SEQ ID NO: 119, SEQ IDNO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124,SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ IDNO: 129, an active vesiculovirus fusogenic peptide domain fragment ofSEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ IDNO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127,SEQ ID NO: 128 or SEQ ID NO: 129, or any combination thereof.

In other aspects of this embodiment, a vesiculovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129, at least 75% amino acididentity with SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129, at least 80% amino acididentity with SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129, at least 85% amino acididentity with SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129, at least 90% amino acididentity with SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129 or at least 95% amino acididentity with SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129. In yet other aspects ofthis embodiment, a vesiculovirus fusogenic peptide domain comprises apolypeptide having, e.g., at most 70% amino acid identity with SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123,SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ IDNO: 128 or SEQ ID NO: 129, at most 75% amino acid identity with SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123,SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ IDNO: 128 or SEQ ID NO: 129, at most 80% amino acid identity with SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123,SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ IDNO: 128 or SEQ ID NO: 129, at most 85% amino acid identity with SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123,SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ IDNO: 128 or SEQ ID NO: 129, at most 90% amino acid identity with SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123,SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ IDNO: 128 or SEQ ID NO: 129 or at most 95% amino acid identity with SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123,SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ IDNO: 128 or SEQ ID NO: 129.

In other aspects of this embodiment, a vesiculovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129. In otheraspects of this embodiment, a vesiculovirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129. In yetother aspects of this embodiment, a vesiculovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino aciddeletions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121,SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ IDNO: 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129. In otheraspects of this embodiment, a vesiculovirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid deletionsrelative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129. In still other aspects ofthis embodiment, a vesiculovirus fusogenic peptide domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toSEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ IDNO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127,SEQ ID NO: 128 or SEQ ID NO: 129. In other aspects of this embodiment, avesiculovirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 non-contiguous amino acid additions relative to SEQ ID NO: 119, SEQID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 orSEQ ID NO: 129.

In other aspects of this embodiment, a vesiculovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129. In otheraspects of this embodiment, a vesiculovirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid substitutionsrelative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129. In yet other aspects ofthis embodiment, a vesiculovirus fusogenic peptide domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid deletions relative to SEQID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQID NO: 128 or SEQ ID NO: 129. In other aspects of this embodiment, avesiculovirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid deletions relative to SEQ ID NO: 119, SEQ IDNO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124,SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ IDNO: 129. In still other aspects of this embodiment, a vesiculovirusfusogenic peptide domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ IDNO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125,SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129. Inother aspects of this embodiment, a vesiculovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidadditions relative to SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121,SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ IDNO: 126, SEQ ID NO: 127, SEQ ID NO: 128 or SEQ ID NO: 129.

In another embodiment, a Clostridial toxin translocation facilitatingdomain comprises a respirovirus fusogenic peptide domain. In anotheraspect of this embodiment, a respirovirus fusogenic peptide domaincomprises a naturally occurring respirovirus fusogenic peptide domainvariant, such as, e.g., a respirovirus fusogenic peptide domain isoformor a respirovirus fusogenic peptide domain subtype. In another aspect ofthis embodiment, a respirovirus fusogenic peptide domain comprises anaturally occurring respirovirus fusogenic peptide domain variant of SEQID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO:134, SEQ ID NO: 135 or SEQ ID NO: 136, such as, e.g., a respirovirusfusogenic peptide domain isoform of SEQ ID NO: 130, SEQ ID NO: 131, SEQID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO:136 or a respirovirus fusogenic peptide domain subtype of SEQ ID NO:130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQID NO: 135 or SEQ ID NO: 136. In still another aspect of thisembodiment, a respirovirus fusogenic peptide domain comprises anon-naturally occurring respirovirus fusogenic peptide domain variant,such as, e.g., a conservative respirovirus fusogenic peptide domainvariant, a non-conservative respirovirus fusogenic peptide domainvariant, a respirovirus fusogenic peptide domain chimeric, an activerespirovirus fusogenic peptide domain fragment, or any combinationthereof. In still another aspect of this embodiment, a respirovirusfusogenic peptide domain comprises amino acids a non-naturally occurringrespirovirus fusogenic peptide domain variant of SEQ ID NO: 130, SEQ IDNO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135or SEQ ID NO: 136, such as, e.g., a conservative respirovirus fusogenicpeptide domain variant of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136, anon-conservative respirovirus fusogenic peptide domain variant of SEQ IDNO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134,SEQ ID NO: 135 or SEQ ID NO: 136, an active respirovirus fusogenicpeptide domain fragment of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136,or any combination thereof.

In other aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO:133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136, at least 75%amino acid identity with SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136, atleast 80% amino acid identity with SEQ ID NO: 130, SEQ ID NO: 131, SEQID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO:136, at least 85% amino acid identity with SEQ ID NO: 130, SEQ ID NO:131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 orSEQ ID NO: 136, at least 90% amino acid identity with SEQ ID NO: 130,SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ IDNO: 135 or SEQ ID NO: 136 or at least 95% amino acid identity with SEQID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO:134, SEQ ID NO: 135 or SEQ ID NO: 136. In yet other aspects of thisembodiment, a respirovirus fusogenic peptide domain comprises apolypeptide having, e.g., at most 70% amino acid identity with SEQ IDNO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134,SEQ ID NO: 135 or SEQ ID NO: 136, at most 75% amino acid identity withSEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ IDNO: 134, SEQ ID NO: 135 or SEQ ID NO: 136, at most 80% amino acididentity with SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO:133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136, at most 85% aminoacid identity with SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136, at most90% amino acid identity with SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136 orat most 95% amino acid identity with SEQ ID NO: 130, SEQ ID NO: 131, SEQID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO:136.

In other aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136.In other aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136.In yet other aspects of this embodiment, a respirovirus fusogenicpeptide domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid deletions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136.In other aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino aciddeletions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136. Instill other aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136. Inother aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidadditions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136.

In other aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136.In other aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136.In yet other aspects of this embodiment, a respirovirus fusogenicpeptide domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136. Inother aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino aciddeletions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136. Instill other aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidadditions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136. Inother aspects of this embodiment, a respirovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidadditions relative to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132,SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 or SEQ ID NO: 136.

In another embodiment, a Clostridial toxin translocation facilitatingdomain comprises a morbillivirus fusogenic peptide domain. In anotheraspect of this embodiment, a morbillivirus fusogenic peptide domaincomprises a naturally occurring morbillivirus fusogenic peptide domainvariant, such as, e.g., a morbillivirus fusogenic peptide domain isoformor a morbillivirus fusogenic peptide domain subtype. In another aspectof this embodiment, a morbillivirus fusogenic peptide domain comprises anaturally occurring morbillivirus fusogenic peptide domain variant ofSEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ IDNO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145or SEQ ID NO: 146, such as, e.g., a morbillivirus fusogenic peptidedomain isoform of SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ IDNO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144,SEQ ID NO: 145 or SEQ ID NO: 146 or a morbillivirus fusogenic peptidedomain subtype of SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ IDNO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144,SEQ ID NO: 145 or SEQ ID NO: 146. In still another aspect of thisembodiment, a morbillivirus fusogenic peptide domain comprises anon-naturally occurring morbillivirus fusogenic peptide domain variant,such as, e.g., a conservative morbillivirus fusogenic peptide domainvariant, a non-conservative morbillivirus fusogenic peptide domainvariant, a morbillivirus fusogenic peptide domain chimeric, an activemorbillivirus fusogenic peptide domain fragment, or any combinationthereof. In still another aspect of this embodiment, a morbillivirusfusogenic peptide domain comprises amino acids a non-naturally occurringmorbillivirus fusogenic peptide domain variant of SEQ ID NO: 137, SEQ IDNO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142,SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146, suchas, e.g., a conservative morbillivirus fusogenic peptide domain variantof SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO:145 or SEQ ID NO: 146, a non-conservative morbillivirus fusogenicpeptide domain variant of SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO:139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146, an active morbillivirusfusogenic peptide domain fragment of SEQ ID NO: 137, SEQ ID NO: 138, SEQID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO:143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146, or anycombination thereof.

In other aspects of this embodiment, a morbillivirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQID NO: 145 or SEQ ID NO: 146, at least 75% amino acid identity with SEQID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO:141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 orSEQ ID NO: 146, at least 80% amino acid identity with SEQ ID NO: 137,SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ IDNO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO:146, at least 85% amino acid identity with SEQ ID NO: 137, SEQ ID NO:138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146, at least90% amino acid identity with SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO:139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146 or at least 95% amino acididentity with SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQID NO: 145 or SEQ ID NO: 146. In yet other aspects of this embodiment, amorbillivirus fusogenic peptide domain comprises a polypeptide having,e.g., at most 70% amino acid identity with SEQ ID NO: 137, SEQ ID NO:138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146, at most75% amino acid identity with SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO:139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146, at most 80% amino acididentity with SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQID NO: 145 or SEQ ID NO: 146, at most 85% amino acid identity with SEQID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO:141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 orSEQ ID NO: 146, at most 90% amino acid identity with SEQ ID NO: 137, SEQID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO:142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146 orat most 95% amino acid identity with SEQ ID NO: 137, SEQ ID NO: 138, SEQID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO:143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146.

In other aspects of this embodiment, a morbillivirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO:139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146. In other aspects of thisembodiment, a morbillivirus fusogenic peptide domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid substitutionsrelative to SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQID NO: 145 or SEQ ID NO: 146. In yet other aspects of this embodiment, amorbillivirus fusogenic peptide domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to SEQ ID NO: 137, SEQ IDNO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142,SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146. Inother aspects of this embodiment, a morbillivirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino aciddeletions relative to SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139,SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ IDNO: 144, SEQ ID NO: 145 or SEQ ID NO: 146. In still other aspects ofthis embodiment, a morbillivirus fusogenic peptide domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toSEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ IDNO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145or SEQ ID NO: 146. In other aspects of this embodiment, a morbillivirusfusogenic peptide domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid additions relative to SEQ ID NO: 137, SEQ IDNO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142,SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146.

In other aspects of this embodiment, a morbillivirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO:139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146. In other aspects of thisembodiment, a morbillivirus fusogenic peptide domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid substitutions relative toSEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ IDNO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145or SEQ ID NO: 146. In yet other aspects of this embodiment, amorbillivirus fusogenic peptide domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to SEQ ID NO: 137, SEQ ID NO:138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146. In otheraspects of this embodiment, a morbillivirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid deletionsrelative to SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQID NO: 145 or SEQ ID NO: 146. In still other aspects of this embodiment,a morbillivirus fusogenic peptide domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid additions relative to SEQ ID NO: 137, SEQ ID NO:138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145 or SEQ ID NO: 146. In otheraspects of this embodiment, a morbillivirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid additionsrelative to SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQID NO: 145 or SEQ ID NO: 146.

In another embodiment, a Clostridial toxin translocation facilitatingdomain comprises an avulavirus fusogenic peptide domain. In anotheraspect of this embodiment, an avulavirus fusogenic peptide domaincomprises a naturally occurring avulavirus fusogenic peptide domainvariant, such as, e.g., an avulavirus fusogenic peptide domain isoformor an avulavirus fusogenic peptide domain subtype. In another aspect ofthis embodiment, an avulavirus fusogenic peptide domain comprises anaturally occurring avulavirus fusogenic peptide domain variant of SEQID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO:151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159, such as,e.g., an avulavirus fusogenic peptide domain isoform of SEQ ID NO: 147,SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ IDNO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156,SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159 or an avulavirusfusogenic peptide domain subtype of SEQ ID NO: 147, SEQ ID NO: 148, SEQID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQID NO: 158 or SEQ ID NO: 159. In still another aspect of thisembodiment, an avulavirus fusogenic peptide domain comprises anon-naturally occurring avulavirus fusogenic peptide domain variant,such as, e.g., a conservative avulavirus fusogenic peptide domainvariant, a non-conservative avulavirus fusogenic peptide domain variant,an avulavirus fusogenic peptide domain chimeric, an active avulavirusfusogenic peptide domain fragment, or any combination thereof. In stillanother aspect of this embodiment, an avulavirus fusogenic peptidedomain comprises amino acids a non-naturally occurring avulavirusfusogenic peptide domain variant of SEQ ID NO: 147, SEQ ID NO: 148, SEQID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQID NO: 158 or SEQ ID NO: 159, such as, e.g., a conservative avulavirusfusogenic peptide domain variant of SEQ ID NO: 147, SEQ ID NO: 148, SEQID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQID NO: 158 or SEQ ID NO: 159, a non-conservative avulavirus fusogenicpeptide domain variant of SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO:158 or SEQ ID NO: 159, an active avulavirus fusogenic peptide domainfragment of SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO:150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO:159, or any combination thereof.

In other aspects of this embodiment, an avulavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO:150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO:159, at least 75% amino acid identity with SEQ ID NO: 147, SEQ ID NO:148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO:157, SEQ ID NO: 158 or SEQ ID NO: 159, at least 80% amino acid identitywith SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO:155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159,at least 85% amino acid identity with SEQ ID NO: 147, SEQ ID NO: 148,SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ IDNO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157,SEQ ID NO: 158 or SEQ ID NO: 159, at least 90% amino acid identity withSEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ IDNO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155,SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159 or atleast 95% amino acid identity with SEQ ID NO: 147, SEQ ID NO: 148, SEQID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQID NO: 158 or SEQ ID NO: 159. In yet other aspects of this embodiment,an avulavirus fusogenic peptide domain comprises a polypeptide having,e.g., at most 70% amino acid identity with SEQ ID NO: 147, SEQ ID NO:148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO:157, SEQ ID NO: 158 or SEQ ID NO: 159, at most 75% amino acid identitywith SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO:155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159,at most 80% amino acid identity with SEQ ID NO: 147, SEQ ID NO: 148, SEQID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQID NO: 158 or SEQ ID NO: 159, at most 85% amino acid identity with SEQID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO:151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159, at most90% amino acid identity with SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO:158 or SEQ ID NO: 159 or at most 95% amino acid identity with SEQ ID NO:147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO:156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159.

In other aspects of this embodiment, an avulavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO:158 or SEQ ID NO: 159. In other aspects of this embodiment, anavulavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 non-contiguous amino acid substitutions relative to SEQ ID NO: 147,SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ IDNO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156,SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159. In yet other aspectsof this embodiment, an avulavirus fusogenic peptide domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toSEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ IDNO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155,SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159. Inother aspects of this embodiment, an avulavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid deletionsrelative to SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO:150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO:159. In still other aspects of this embodiment, an avulavirus fusogenicpeptide domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or 10 non-contiguous aminoacid additions relative to SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO:158 or SEQ ID NO: 159. In other aspects of this embodiment, anavulavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 non-contiguous amino acid additions relative to SEQ ID NO: 147, SEQID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159.

In other aspects of this embodiment, an avulavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO:158 or SEQ ID NO: 159. In other aspects of this embodiment, anavulavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid substitutions relative to SEQ ID NO: 147, SEQID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159. In yet other aspects ofthis embodiment, an avulavirus fusogenic peptide domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid deletions relative to SEQID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO:151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO: 159. In otheraspects of this embodiment, an avulavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid deletionsrelative to SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO:150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158 or SEQ ID NO:159. In still other aspects of this embodiment, an avulavirus fusogenicpeptide domain comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or 10 contiguous amino acidadditions relative to SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149,SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ IDNO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158or SEQ ID NO: 159. In other aspects of this embodiment, an avulavirusfusogenic peptide domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to SEQ ID NO: 147, SEQ ID NO: 148, SEQ IDNO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153,SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ IDNO: 158 or SEQ ID NO: 159.

In another embodiment, a Clostridial toxin translocation facilitatingdomain comprises a henipavirus fusogenic peptide domain. In anotheraspect of this embodiment, a henipavirus fusogenic peptide domaincomprises a naturally occurring henipavirus fusogenic peptide domainvariant, such as, e.g., a henipavirus fusogenic peptide domain isoformor a henipavirus fusogenic peptide domain subtype. In another aspect ofthis embodiment, a henipavirus fusogenic peptide domain comprises anaturally occurring henipavirus fusogenic peptide domain variant of SEQID NO: 160 or SEQ ID NO: 161, such as, e.g., a henipavirus fusogenicpeptide domain isoform of SEQ ID NO: 160 or SEQ ID NO: 161 or ahenipavirus fusogenic peptide domain subtype of SEQ ID NO: 160 or SEQ IDNO: 161. In still another aspect of this embodiment, a henipavirusfusogenic peptide domain comprises a non-naturally occurring henipavirusfusogenic peptide domain variant, such as, e.g., a conservativehenipavirus fusogenic peptide domain variant, a non-conservativehenipavirus fusogenic peptide domain variant, a henipavirus fusogenicpeptide domain chimeric, an active henipavirus fusogenic peptide domainfragment, or any combination thereof. In still another aspect of thisembodiment, a henipavirus fusogenic peptide domain comprises amino acidsa non-naturally occurring henipavirus fusogenic peptide domain variantof SEQ ID NO: 160 or SEQ ID NO: 161, such as, e.g., a conservativehenipavirus fusogenic peptide domain variant of SEQ ID NO: 160 or SEQ IDNO: 161, a non-conservative henipavirus fusogenic peptide domain variantof SEQ ID NO: 160 or SEQ ID NO: 161, an active henipavirus fusogenicpeptide domain fragment of SEQ ID NO: 160 or SEQ ID NO: 161, or anycombination thereof.

In other aspects of this embodiment, a henipavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 160 or SEQ ID NO: 161, at least 75% amino acididentity with SEQ ID NO: 160 or SEQ ID NO: 161, at least 80% amino acididentity with SEQ ID NO: 160 or SEQ ID NO: 161, at least 85% amino acididentity with SEQ ID NO: 160 or SEQ ID NO: 161, at least 90% amino acididentity with SEQ ID NO: 160 or SEQ ID NO: 161 or at least 95% aminoacid identity with SEQ ID NO: 160 or SEQ ID NO: 161. In yet otheraspects of this embodiment, a henipavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at most 70% amino acid identitywith SEQ ID NO: 160 or SEQ ID NO: 161, at most 75% amino acid identitywith SEQ ID NO: 160 or SEQ ID NO: 161, at most 80% amino acid identitywith SEQ ID NO: 160 or SEQ ID NO: 161, at most 85% amino acid identitywith SEQ ID NO: 160 or SEQ ID NO: 161, at most 90% amino acid identitywith SEQ ID NO: 160 or SEQ ID NO: 161 or at most 95% amino acid identitywith SEQ ID NO: 160 or SEQ ID NO: 161.

In other aspects of this embodiment, a henipavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 160 or SEQ ID NO: 161. In otheraspects of this embodiment, a henipavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 160 or SEQ ID NO: 161. In yet otheraspects of this embodiment, a henipavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid deletionsrelative to SEQ ID NO: 160 or SEQ ID NO: 161. In other aspects of thisembodiment, a henipavirus fusogenic peptide domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toSEQ ID NO: 160 or SEQ ID NO: 161. In still other aspects of thisembodiment, a henipavirus fusogenic peptide domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid additions relative toSEQ ID NO: 160 or SEQ ID NO: 161. In other aspects of this embodiment, ahenipavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 non-contiguous amino acid additions relative to SEQ ID NO: 160 or SEQID NO: 161.

In other aspects of this embodiment, a henipavirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 160 or SEQ ID NO: 161. In otheraspects of this embodiment, a henipavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid substitutionsrelative to SEQ ID NO: 160 or SEQ ID NO: 161. In yet other aspects ofthis embodiment, a henipavirus fusogenic peptide domain comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid deletions relative to SEQID NO: 160 or SEQ ID NO: 161. In other aspects of this embodiment, ahenipavirus fusogenic peptide domain comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid deletions relative to SEQ ID NO: 160 or SEQ IDNO: 161. In still other aspects of this embodiment, a henipavirusfusogenic peptide domain comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid additions relative to SEQ ID NO: 160 or SEQ ID NO: 161. Inother aspects of this embodiment, a henipavirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid additionsrelative to SEQ ID NO: 160 or SEQ ID NO: 161.

In another embodiment, a Clostridial toxin translocation facilitatingdomain comprises a metapneumovirus fusogenic peptide domain. In anotheraspect of this embodiment, a metapneumovirus fusogenic peptide domaincomprises a naturally occurring metapneumovirus fusogenic peptide domainvariant, such as, e.g., a metapneumovirus fusogenic peptide domainisoform or a metapneumovirus fusogenic peptide domain subtype. Inanother aspect of this embodiment, a metapneumovirus fusogenic peptidedomain comprises a naturally occurring metapneumovirus fusogenic peptidedomain variant of SEQ ID NO: 162, such as, e.g., a metapneumovirusfusogenic peptide domain isoform of SEQ ID NO: 162 or a metapneumovirusfusogenic peptide domain subtype of SEQ ID NO: 162. In still anotheraspect of this embodiment, a metapneumovirus fusogenic peptide domaincomprises a non-naturally occurring metapneumovirus fusogenic peptidedomain variant, such as, e.g., a conservative metapneumovirus fusogenicpeptide domain variant, a non-conservative metapneumovirus fusogenicpeptide domain variant, a metapneumovirus fusogenic peptide domainchimeric, an active metapneumovirus fusogenic peptide domain fragment,or any combination thereof. In still another aspect of this embodiment,a metapneumovirus fusogenic peptide domain comprises amino acids anon-naturally occurring metapneumovirus fusogenic peptide domain variantof SEQ ID NO: 162, such as, e.g., a conservative metapneumovirusfusogenic peptide domain variant of SEQ ID NO: 162, a non-conservativemetapneumovirus fusogenic peptide domain variant of SEQ ID NO: 162, anactive metapneumovirus fusogenic peptide domain fragment of SEQ ID NO:162, or any combination thereof.

In other aspects of this embodiment, a metapneumovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 162, at least 75% amino acid identity with SEQID NO: 162, at least 80% amino acid identity with SEQ ID NO: 162, atleast 85% amino acid identity with SEQ ID NO: 162, at least 90% aminoacid identity with SEQ ID NO: 162 or at least 95% amino acid identitywith SEQ ID NO: 162. In yet other aspects of this embodiment, ametapneumovirus fusogenic peptide domain comprises a polypeptide having,e.g., at most 70% amino acid identity with SEQ ID NO: 162, at most 75%amino acid identity with SEQ ID NO: 162, at most 80% amino acid identitywith SEQ ID NO: 162, at most 85% amino acid identity with SEQ ID NO:162, at most 90% amino acid identity with SEQ ID NO: 162 or at most 95%amino acid identity with SEQ ID NO: 162.

In other aspects of this embodiment, a metapneumovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 162. In other aspects of thisembodiment, a metapneumovirus fusogenic peptide domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid substitutionsrelative to SEQ ID NO: 162. In yet other aspects of this embodiment, ametapneumovirus fusogenic peptide domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to SEQ ID NO: 162. In otheraspects of this embodiment, a metapneumovirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid deletionsrelative to SEQ ID NO: 162. In still other aspects of this embodiment, ametapneumovirus fusogenic peptide domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid additions relative to SEQ ID NO: 162. In otheraspects of this embodiment, a metapneumovirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to SEQ ID NO: 162.

In other aspects of this embodiment, a metapneumovirus fusogenic peptidedomain comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to SEQ ID NO: 162. In other aspects of thisembodiment, a metapneumovirus fusogenic peptide domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid substitutions relative toSEQ ID NO: 162. In yet other aspects of this embodiment, ametapneumovirus fusogenic peptide domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid deletions relative to SEQ ID NO: 162. In otheraspects of this embodiment, a metapneumovirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid deletionsrelative to SEQ ID NO: 162. In still other aspects of this embodiment, ametapneumovirus fusogenic peptide domain comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10contiguous amino acid additions relative to SEQ ID NO: 162. In otheraspects of this embodiment, a metapneumovirus fusogenic peptide domaincomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid additionsrelative to SEQ ID NO: 162.

Aspects of the present invention provide, in part, an altered targetingdomain. As used herein, the term “altered targeting domain” means anypolypeptide that can selectively bind to a non-Clostridial toxinreceptor present on a non-Clostridial toxin target cell and initiate theoverall internalization mechanism whereby the modified Clostridial toxindisclosed in the present specification intoxicates a target cell. Asused herein, the term “selectively” means having a highly preferredactivity or effect. As used herein, the term “selectively bind” means amolecule is able to bind its target receptor under physiologicalconditions, or in vitro conditions substantially approximatingphysiological conditions, to a statistically significantly greaterdegree relative to other, non-target receptors. Thus, with reference toan altered targeting domain of the present specification, there is adiscriminatory binding of the altered targeting domain to anon-Clostridial toxin receptor presence in a non-Clostridial toxintarget cell.

Aspects of the present invention provide, in part, an enhanced targetingdomain. As used herein, the term “enhanced targeting domain” means anypolypeptide that can selectively bind to an endogenous Clostridial toxinreceptor found on a Clostridial toxin target cell and initiate theoverall internalization mechanism whereby the modified Clostridial toxindisclosed in the present specification intoxicates a target cell withthe proviso that an enhanced targeting domain is not anaturally-occurring binding domain from a naturally occurringClostridial toxin. As used herein, the term “selectively” means having ahighly preferred activity or effect. As used herein, the term“selectively bind” means a molecule is able to bind its target receptorunder physiological conditions, or in vitro conditions substantiallyapproximating physiological conditions, to a statistically significantlygreater degree relative to other non-target receptors. Thus, withreference to an enhanced targeting domain of the present specification,there is a discriminatory binding of the enhanced targeting domain to anendogenous Clostridial toxin receptor.

An enhanced targeting domain disclosed in the present specificationfacilitates the binding activity of the modified Clostridial toxinsdisclosed in the present specification to an endogenous Clostridialtoxin receptor located at the surface of a Clostridial toxin targetcell. As used herein, the term “binding activity” means that onemolecule is directly or indirectly contacting another molecule via atleast one intermolecular or intramolecular force, including, withoutlimitation, a covalent bond, an ionic bond, a metallic bond, a hydrogenbond, a hydrophobic interaction, a van der Waals interaction, and thelike, or any combination thereof. “Bound” and “bind” are consideredterms for binding.

As used herein, the term “binding affinity” means how strong amolecule's binding activity is for a particular receptor. In general,high binding affinity results from greater intermolecular force betweena binding domain and its receptor while low binding affinity involvesless intermolecular force between the ligand and its receptor. Highbinding affinity involves a longer residence time for the binding domainat its receptor binding site than is the case for low binding affinity.As such, a molecule with a high binding affinity means a lowerconcentration of that molecule is required to maximally occupy thebinding sites of a receptor and trigger a physiological response.Conversely, low binding affinity means a relatively high concentrationof a molecule is required before the receptor binding sites of areceptor is maximally occupied and the maximum physiological response isachieved. Thus, modified Clostridial toxins with increased bindingactivity due to high binding affinity will allow administration ofreduced doses of the toxin, thereby reducing or preventing unwantedside-effects associated with toxin dispersal into non-targeted areas.

As used herein, the term “binding specificity” means how specific amolecule's binding activity is one particular receptor. In general, highbinding specificity results in a more exclusive interaction with oneparticular receptor or subgroup of receptors while low bindingspecificity results in a more promiscuous interaction with a largergroup of receptors. As such, a molecule with a high binding specificitymeans that molecule will occupy the binding sites of a particularreceptor and trigger a physiological response. Conversely, low bindingspecificity means a molecule will occupy the binding sites of manyreceptors and trigger a multitude of physiological responses. Thus,modified Clostridial toxins with increased binding activity due to highbinding specificity will only target a subgroup of Clostridial toxintarget cells, thereby reducing the side effects associated with thetargeting of all Clostridial toxin target cells.

It is envisioned that any and all enhanced targeting domains can be usedto practice aspects of the present invention, including, withoutlimitation, an enhanced targeting domain that increases binding affinityfor an endogenous Clostridial toxin receptor present on anaturally-occurring Clostridial toxin target cell; and an enhancedtargeting domain that increases binding specificity for a subgroup ofendogenous Clostridial toxin receptors present on a naturally-occurringClostridial toxin target cell.

Assays that determine modified Clostridial toxin binding activity oruptake properties can be used to assess whether a modified Clostridialtoxin disclosed in the present specification has an enhanced bindingactivity, such as an increased binding affinity or an increased bindingspecificity. It is envisioned that heterogeneous assay types,homogeneous assay types and non-separating homogeneous assay types canbe used to determine the binding activity of a modified Clostridialtoxin with an enhanced targeting domain, see, e.g., Lutea A. A. de Jonget al., Receptor-Binding Assays: Technologies and Applications, 829 J.Chromatogr. B 1-25 (2005). It is further envisioned that the bindingactivity of a modified Clostridial toxin with an enhanced targetingdomain can be determined by affinity chromatography using immobilizedreceptors and interfacial optical assays, such as, e.g., total internalreflection fluorescence (TIRF) and surface plasmon resonance (SPR).Non-limiting examples of these assays include, e.g., FCS using diffusionmediated intensity fluctuations, SPR using a mass-dependent refractiveindex, TIRF using a mass-independent refractive index, microarrays usingoptical intensity changes and QAC using retention volume.

As a non-limiting example, cross-linking assays using radio-labeled ornon-radio-labeled modified Clostridial toxins can determine whether sucha modified toxin has an enhanced targeting activity to a Clostridialtoxin receptor as compared to the Clostridial toxin from which themodified toxin was derived. Other non-limiting assays includeimmunocytochemical assays that detect modified toxin binding usinglabeled or unlabeled antibodies and immunoprecipitation assays thatdetect bound modified toxin using labeled or unlabeled antibodies.Antibodies useful for these assays include, without limitation,antibodies selected against a portion of a Clostridial toxin heavy chaincomprising the H_(N) translocation domain or a portion of a Clostridialtoxin light chain; or antibodies selected against the Clostridial toxinreceptor. If the antibody is labeled, the binding of the molecule can bedetected by various means, including Western blotting, directmicroscopic observation of the cellular location of the antibody,measurement of cell or substrate-bound antibody following a wash step,or electrophoresis, employing techniques well-known to those of skill inthe art. If the antibody is unlabeled, one may employ a labeledsecondary antibody for indirect detection of the bound molecule, anddetection can proceed as for a labeled antibody. It is understood thatthese and similar assays that determine modified Clostridial toxinuptake properties or characteristics can be useful in determiningwhether a modified toxin disclosed in the present specification has anenhanced targeting activity.

Assays that monitor the release of a molecule after exposure to amodified Clostridial toxin disclosed in the present specification canalso be used to assess whether such a modified toxin has enhancedbinding activity to a Clostridial toxin receptor as compared to theClostridial toxin from which the modified toxin was derived. In theseassays, a greater or faster inhibition of the molecule's release wouldoccur in cells exposed to a modified Clostridial toxin with an enhancedtargeting activity after exposure to a modified Clostridial toxin. Wellknown assays include methods that measure inhibition of radio-labeledcatecholamine release from neurons, such as, e.g., [³H]noradrenaline or[³H]dopamine release, see e.g., A Fassio et al., Evidence forcalcium-dependent vesicular transmitter release insensitive to tetanustoxin and botulinum toxin type F, 90(3) Neuroscience 893-902 (1999); andSara Stigliani et al., The sensitivity of catecholamine release tobotulinum toxin C1 and E suggests selective targeting of vesicles setinto the readily releasable pool, 85(2) J. Neurochem. 409-421 (2003), ormeasures catecholamine release using a fluorometric procedure, see,e.g., Anton de Paiva et al., A role for the interchain disulfide or itsparticipating thiols in the internalization of botulinum neurotoxin Arevealed by a toxin derivative that binds to ecto-acceptors and inhibitstransmitter release intracellularly, 268(28) J. Biol. Chem. 20838-20844(1993); Gary W. Lawrence et al., Distinct exocytotic responses of intactand permeabilised chromaffin cells after cleavage of the 25-kDasynaptosomal-associated protein (SNAP-25) or synaptobrevin by botulinumtoxin A or B, 236(3) Eur. J. Biochem. 877-886 (1996); and Patrick Foranet al., Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 inintact and permeabilized chromaffin cells: correlation with its blockadeof catecholamine release, 35(8) Biochemistry 2630-2636 (1996). Othernon-limiting examples include assays that measure inhibition of hormonerelease from endocrine cells, such as, e.g., anterior pituitary cells orovarian cells. It is understood that these and similar assays formolecule release can be useful in determining whether a modified toxindisclosed in the present specification has an enhanced targetingactivity.

Assays that detect the cleavage of a Clostridial toxin substrate afterexposure to a modified Clostridial toxin disclosed in the presentspecification can also be used to assess whether such a modified toxinhas enhanced binding activity to a Clostridial toxin receptor ascompared to the Clostridial toxin from which the modified toxin wasderived. In these assays, generation of a Clostridial toxin substratecleavage-product would be detected in cells expressing a receptor ofinterest after modified Clostridial toxin treatment. Non-limitingexamples of specific Western blotting procedures, as well aswell-characterized reagents, conditions and protocols are readilyavailable from commercial vendors that include, without limitation,Amersham Biosciences, Piscataway, N.J.; Bio-Rad Laboratories, Hercules,Calif.; Pierce Biotechnology, Inc., Rockford, Ill.; Promega Corporation,Madison, Wis., and Stratagene, Inc., La Jolla, Calif. It is understoodthat these and similar assays for Clostridial toxin substrate cleavagecan be useful in determining whether a modified toxin disclosed in thepresent specification has an enhanced targeting activity.

As non-limiting examples, western blot analysis using an antibody thatspecifically recognizes a BoNT/A substrate cleavage product at theSNAP-25 site can be used to assay whether a modified toxin has anenhanced binding activity to a Clostridial toxin receptor as compared tothe Clostridial toxin from which the modified toxin was derived; westernblot analysis using an antibody that specifically recognizes a BoNT/C1substrate cleavage product at the SNAP-25 site can be used to assaywhether a modified toxin has an enhanced binding activity to aClostridial toxin receptor as compared to the Clostridial toxin fromwhich the modified toxin was derived; and western blot analysis using anantibody that specifically recognizes a BoNT/E substrate cleaved productcan be used to assay whether a modified toxin has an enhanced bindingactivity to a Clostridial toxin receptor as compared to the Clostridialtoxin from which the modified toxin was derived. Examples ofanti-SNAP-25 antibodies useful for these assays include, withoutlimitation, rabbit polyclonal anti-SNAP25₁₉₇ antiserum pAbanti-SNAP25197 #1 (Allergan, Inc., Irvine, Calif.), mouse monoclonalanti-SNAP-25 antibody SMI-81 (Sternberger Monoclonals, Lutherville,Md.), mouse monoclonal anti-SNAP-25 antibody CI 71.1 (Synaptic Systems,Goettingen, Germany), mouse monoclonal anti-SNAP-25 antibody CI 71.2(Synaptic Systems, Goettingen, Germany), mouse monoclonal anti-SNAP-25antibody SP12 (Abcam, Cambridge, Mass.), rabbit polyclonal anti-SNAP-25antiserum (Synaptic Systems, Goettingen, Germany), and rabbit polyclonalanti-SNAP-25 antiserum (Abcam, Cambridge, Mass.).

As additional non-limiting examples, western blot analysis using anantibody that specifically recognizes a BoNT/B substrate cleaved productcan be used to assay whether a modified toxin has an enhanced bindingactivity to a Clostridial toxin receptor as compared to the Clostridialtoxin from which the modified toxin was derived; western blot analysisusing an antibody that specifically recognizes a BoNT/D substratecleaved product can be used to assay whether a modified toxin has anenhanced binding activity to a Clostridial toxin receptor as compared tothe Clostridial toxin from which the modified toxin was derived; westernblot analysis using an antibody that specifically recognizes a BoNT/Fsubstrate cleaved product can be used to assay whether a modified toxinhas an enhanced binding activity to a Clostridial toxin receptor ascompared to the Clostridial toxin from which the modified toxin wasderived; western blot analysis using an antibody that specificallyrecognizes a BoNT/G substrate cleaved product can be used to assaywhether a modified toxin has an enhanced binding activity to aClostridial toxin receptor as compared to the Clostridial toxin fromwhich the modified toxin was derived; and western blot analysis using anantibody that specifically recognizes a TeNT substrate cleaved productcan be used to assay whether a modified toxin has an enhanced bindingactivity to a Clostridial toxin receptor as compared to the Clostridialtoxin from which the modified toxin was derived. Examples of anti-VAMPantibodies useful for these assays include, without limitation, mousemonoclonal anti-VAMP-1 antibody CI 10.1 (Synaptic Systems, Goettingen,Germany), mouse monoclonal anti-VAMP-1 antibody SP10 (Abcam, Cambridge,Mass.), mouse monoclonal anti-VAMP-1 antibody SP11 (Abcam, Cambridge,Mass.), rabbit polyclonal anti-VAMP-1 antiserum (Synaptic Systems,Goettingen, Germany), rabbit polyclonal anti-VAMP-1 antiserum (Abcam,Cambridge, Mass.), mouse monoclonal anti-VAMP-2 antibody CI 69.1(Synaptic Systems, Goettingen, Germany), rabbit polyclonal anti-VAMP-2antiserum (Synaptic Systems, Goettingen, Germany), rabbit polyclonalanti-VAMP-2 antiserum (Abcam, Cambridge, Mass.), mouse monoclonalanti-VAMP-3 antibody CI 10.1 (Synaptic Systems, Goettingen, Germany),rabbit polyclonal anti-VAMP-3 antiserum (Synaptic Systems, Goettingen,Germany) and rabbit polyclonal anti-VAMP-3 antiserum (Abcam, Cambridge,Mass.),

As another non-limiting example, western blot analysis using an antibodythat specifically recognizes a BoNT/C1 substrate cleaved product at theSyntaxin site can be used to assay whether a modified toxin has anenhanced binding activity to a Clostridial toxin receptor as compared tothe Clostridial toxin from which the modified toxin was derived.Examples of anti-Syntaxin antibodies useful for these assays include,without limitation, mouse monoclonal anti-Syntaxin-1 antibody CI 78.2(Synaptic Systems, Goettingen, Germany), mouse monoclonalanti-Syntaxin-1A antibody CI 78.3 (Synaptic Systems, Goettingen,Germany), rabbit polyclonal anti-Syntaxin-1A antiserum (SynapticSystems, Goettingen, Germany), rabbit polyclonal anti-Syntaxin-1Bantiserum (Synaptic Systems, Goettingen, Germany), rabbit polyclonalanti-Syntaxin antiserum (Abcam, Cambridge, Mass.), rabbit polyclonalanti-Syntaxin-2 antiserum (Abcam, Cambridge, Mass.) and rabbitpolyclonal anti-Syntaxin-3 antiserum (Abcam, Cambridge, Mass.),

Thus, in an embodiment, a modified Clostridial toxin with an enhancedtargeting activity is a modified Clostridial toxin with an increasedbinding affinity for a Clostridial toxin receptor. Increased bindingaffinity for a Clostridial toxin receptor is determined by comparing thebinding affinity of the modified Clostridial toxin to that of thebinding affinity for the same Clostridial toxin receptor of aClostridial toxin lacking the enhanced targeting domain modificationfrom which the modified Clostridial toxin is derived. In aspects of thisembodiment, a modified Clostridial toxin with an increased bindingaffinity for a Clostridial toxin receptor exhibits a binding affinitythat is, e.g., at least 10% greater than the binding affinity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived, at least 20% greater than the bindingaffinity of the naturally-occurring Clostridial toxin from which themodified Clostridial toxin is derived, at least 30% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at least 40% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at least 50% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at least 60% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at least 70% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at least 80% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at least 90% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived or at least 100% greater thanthe binding affinity of the naturally-occurring Clostridial toxin fromwhich the modified Clostridial toxin is derived. In aspects of thisembodiment, a modified Clostridial toxin with an increased bindingaffinity for a Clostridial toxin receptor exhibits a binding affinitythat is, e.g., at most 10% greater than the binding affinity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived, at most 20% greater than the bindingaffinity of the naturally-occurring Clostridial toxin from which themodified Clostridial toxin is derived, at most 30% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at most 40% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at most 50% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at most 60% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at most 70% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at most 80% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at most 90% greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived or at most 100% greater thanthe binding affinity of the naturally-occurring Clostridial toxin fromwhich the modified Clostridial toxin is derived.

In yet other aspects of this embodiment, a modified Clostridial toxinwith an increased binding affinity for a Clostridial toxin receptorexhibits a binding affinity that is, e.g., at least two-fold greaterthan the binding affinity of the naturally-occurring Clostridial toxinfrom which the modified Clostridial toxin is derived, at leastthree-fold greater than the binding affinity of the naturally-occurringClostridial toxin from which the modified Clostridial toxin is derived,at least four-fold greater than the binding affinity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived, at least five-fold greater than thebinding affinity of the naturally-occurring Clostridial toxin from whichthe modified Clostridial toxin is derived, at least ten-fold greaterthan the binding affinity of the naturally-occurring Clostridial toxinfrom which the modified Clostridial toxin is derived or at leasttwenty-fold greater than the binding affinity of the naturally-occurringClostridial toxin from which the modified Clostridial toxin is derived.In yet other aspects of this embodiment, a modified Clostridial toxinwith an increased binding affinity for a Clostridial toxin receptorexhibits a binding affinity that is, e.g., at most two-fold greater thanthe binding affinity of the naturally-occurring Clostridial toxin fromwhich the modified Clostridial toxin is derived, at most three-foldgreater than the binding affinity of the naturally-occurring Clostridialtoxin from which the modified Clostridial toxin is derived, at mostfour-fold greater than the binding affinity of the naturally-occurringClostridial toxin from which the modified Clostridial toxin is derived,at most five-fold greater than the binding affinity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived, at most ten-fold greater than the bindingaffinity of the naturally-occurring Clostridial toxin from which themodified Clostridial toxin is derived or at most twenty-fold greaterthan the binding affinity of the naturally-occurring Clostridial toxinfrom which the modified Clostridial toxin is derived.

In another embodiment, a modified Clostridial toxin with an enhancedtargeting activity is a modified Clostridial toxin with an increasedbinding specificity for a Clostridial toxin receptor. Increased bindingspecificity for a Clostridial toxin receptor is determined by comparingthe binding specificity of the modified Clostridial toxin to that of thebinding specificity for the same Clostridial toxin receptor of aClostridial toxin lacking the enhanced targeting domain modificationfrom which the modified Clostridial toxin is derived. In aspects of thisembodiment, a modified Clostridial toxin with an increased bindingspecificity for a Clostridial toxin receptor exhibits a bindingspecificity that is, e.g., at least 10% greater than the bindingspecificity of the naturally-occurring Clostridial toxin from which themodified Clostridial toxin is derived, at least 20% greater than thebinding specificity of the naturally-occurring Clostridial toxin fromwhich the modified Clostridial toxin is derived, at least 30% greaterthan the binding specificity of the naturally-occurring Clostridialtoxin from which the modified Clostridial toxin is derived, at least 40%greater than the binding specificity of the naturally-occurringClostridial toxin from which the modified Clostridial toxin is derived,at least 50% greater than the binding specificity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived, at least 60% greater than the bindingspecificity of the naturally-occurring Clostridial toxin from which themodified Clostridial toxin is derived, at least 70% greater than thebinding specificity of the naturally-occurring Clostridial toxin fromwhich the modified Clostridial toxin is derived, at least 80% greaterthan the binding specificity of the naturally-occurring Clostridialtoxin from which the modified Clostridial toxin is derived, at least 90%greater than the binding specificity of the naturally-occurringClostridial toxin from which the modified Clostridial toxin is derivedor at least 100% greater than the binding specificity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived. In aspects of this embodiment, a modifiedClostridial toxin with an increased binding specificity for aClostridial toxin receptor exhibits a binding specificity that is, e.g.,at most 10% greater than the binding specificity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived, at most 20% greater than the bindingspecificity of the naturally-occurring Clostridial toxin from which themodified Clostridial toxin is derived, at most 30% greater than thebinding specificity of the naturally-occurring Clostridial toxin fromwhich the modified Clostridial toxin is derived, at most 40% greaterthan the binding specificity of the naturally-occurring Clostridialtoxin from which the modified Clostridial toxin is derived, at most 50%greater than the binding specificity of the naturally-occurringClostridial toxin from which the modified Clostridial toxin is derived,at most 60% greater than the binding specificity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived, at most 70% greater than the bindingspecificity of the naturally-occurring Clostridial toxin from which themodified Clostridial toxin is derived, at most 80% greater than thebinding specificity of the naturally-occurring Clostridial toxin fromwhich the modified Clostridial toxin is derived, at most 90% greaterthan the binding specificity of the naturally-occurring Clostridialtoxin from which the modified Clostridial toxin is derived or at most100% greater than the binding specificity of the naturally-occurringClostridial toxin from which the modified Clostridial toxin is derived.

In yet other aspects of this embodiment, a modified Clostridial toxinwith an increased binding specificity for a Clostridial toxin receptorexhibits a binding specificity that is, e.g., at least two-fold greaterthan the binding specificity of the naturally-occurring Clostridialtoxin from which the modified Clostridial toxin is derived, at leastthree-fold greater than the binding specificity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived, at least four-fold greater than thebinding specificity of the naturally-occurring Clostridial toxin fromwhich the modified Clostridial toxin is derived, at least five-foldgreater than the binding specificity of the naturally-occurringClostridial toxin from which the modified Clostridial toxin is derived,at least ten-fold greater than the binding specificity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived or at least twenty-fold greater than thebinding specificity of the naturally-occurring Clostridial toxin fromwhich the modified Clostridial toxin is derived. In yet other aspects ofthis embodiment, a modified Clostridial toxin with an increased bindingspecificity for a Clostridial toxin receptor exhibits a bindingspecificity that is, e.g., at most two-fold greater than the bindingspecificity of the naturally-occurring Clostridial toxin from which themodified Clostridial toxin is derived, at most three-fold greater thanthe binding specificity of the naturally-occurring Clostridial toxinfrom which the modified Clostridial toxin is derived, at most four-foldgreater than the binding specificity of the naturally-occurringClostridial toxin from which the modified Clostridial toxin is derived,at most five-fold greater than the binding specificity of thenaturally-occurring Clostridial toxin from which the modifiedClostridial toxin is derived, at most ten-fold greater than the bindingspecificity of the naturally-occurring Clostridial toxin from which themodified Clostridial toxin is derived or at most twenty-fold greaterthan the binding specificity of the naturally-occurring Clostridialtoxin from which the modified Clostridial toxin is derived.

As used herein, the term “Clostridial toxin target cell” means a cellthat is a naturally occurring cell that a naturally occurringClostridial toxin is capable of intoxicating, including, withoutlimitation, motor neurons; sensory neurons; autonomic neurons; such as,e.g., sympathetic neurons and parasympathetic neurons; non-peptidergicneurons, such as, e.g., cholinergic neurons, adrenergic neurons,noradrenergic neurons, serotonergic neurons, GABAergic neurons; andpeptidergic neurons, such as, e.g., Substance P neurons, Calcitonin GeneRelated Peptide neurons, vasoactive intestinal peptide neurons,Neuropeptide Y neurons, cholecystokinin neurons.

An enhanced targeting domain disclosed in the present specificationreplaces the binding activity of the Clostridial toxin targeting domainfound in naturally occurring Clostridial toxins. As used herein, theterm “naturally occurring Clostridial toxin targeting domain” issynonymous with “naturally occurring Clostridial toxin H_(CC) targetingdomain” and means any naturally occurring Clostridial toxin polypeptidethat can execute the cell binding step of the intoxication process,including, e.g., the binding of the Clostridial toxin to a Clostridialtoxin-specific receptor located on the plasma membrane surface of atarget cell. It is envisioned that replacement of the binding activityof a naturally occurring Clostridial toxin H_(CC) targeting domain by anenhanced targeting domain can be achieved by, e.g., replacing the entirenaturally occurring Clostridial toxin H_(CC) targeting domain with anenhanced targeting domain; replacing a portion of a naturally occurringClostridial toxin H_(CC) targeting domain with an enhanced targetingdomain; and operably-linking an enhanced targeting domain to a naturallyoccurring Clostridial toxin comprising a Clostridial toxin H_(CC)targeting domain.

An example of an enhanced targeting domain that increases bindingactivity for an endogenous Clostridial toxin receptor present on anaturally-occurring Clostridial toxin target cell, includes, withoutlimitation, a modified Clostridial toxin H_(CC) targeting domain withenhanced binding activity, such as, e.g., a modified BoNT/A H_(CC)targeting domain with enhanced binding activity, a modified BoNT/BH_(CC) targeting domain with enhanced binding activity, a modifiedBoNT/C1 H_(CC) targeting domain with enhanced binding activity, amodified BoNT/D H_(CC) targeting domain with enhanced binding activity,a modified BoNT/E H_(CC) targeting domain with enhanced bindingactivity, a modified BoNT/F H_(CC) targeting domain with enhancedbinding activity, a modified BoNT/G H_(CC) targeting domain withenhanced binding activity and a modified TeNT H_(CC) targeting domainwith enhanced binding activity. Examples of a modified Clostridial toxinH_(CC) targeting domain that increase binding activity include, e.g., amodified Clostridial toxin H_(CC) targeting domain that increasesbinding affinity for an endogenous Clostridial toxin receptor present ona naturally-occurring Clostridial toxin target cell and a modifiedClostridial toxin H_(CC) targeting domain that increases bindingspecificity for a subgroup of endogenous Clostridial toxin receptorspresent on a naturally-occurring Clostridial toxin target cell.

The three-dimensional crystal structures of BoNT/A, BoNT/B and the H_(C)domain of TeNT indicate that the carboxyl-terminal H_(CC) domaincomprises a modified β-trefoil domain which forms three distinctcarbohydrate binding regions that resembles the carbohydrate bindingmoiety found in many sugar-binding proteins, such as, e.g., serumamyloid P, sialidase, cryia, insecticidal α-endotoxin and lectins.Biochemical studies indicate that the β-trefoil domain structure of theH_(CC) domain appears to mediate the binding to specific carbohydratecontaining components of the Clostridial toxin receptor on the cellsurface, see, e.g., Krzysztof Ginalski et al., Structure-based SequenceAlignment for the Beta-Trefoil Subdomain of the Clostridial NeurotoxinFamily Provides Residue Level Information About the Putative GangliosideBinding Site, 482(1-2) FEBS Lett. 119-124 (2000).

Proteins containing the structural β-trefoil domain represents a diversegroup of proteins, see, e.g., C. A. Orengo et al., Protein Superfamiliesand Domain Superfolds, 372 Nature 631-634 (1994). The β-trefoil domaincomprises a six-stranded β-barrel closed off at one end by threeβ-hairpin structures that exhibits a characteristic pseudo-threefoldaxis symmetry. The monomeric structural unit of this three-fold symmetryis referred to as the β-trefoil fold that contains four β-sheetsorganized as a pair of antiparallel β-sheets. Dividing each of theseβ-trefoil folds is a β-hairpin turn. Therefore, in a linear fashion, aβ-trefoil domain comprises four β-sheets of the first β-trefoil fold(α-fold), a β-hairpin turn, four β-sheets of the second β-trefoil fold(β-fold), a second β-hairpin turn four β-sheets of the third β-trefoilfold (γ-fold)(see FIG. 2). Because the first hairpin turn is locatedbetween the fourth and fifth β-sheets of the β-trefoil domain, it isdesignated the β4/β5 β-hairpin turn. Likewise, since the second hairpinturn is located between the eight and ninth β-sheets of the β-trefoildomain, it is designated the β8/β9 β-hairpin turn.

Continuing research has elucidated that β4/β5 and β8/β9 β-hairpin turnsare important in conferring the proper pseudo-threefold axis symmetryobserved in the β-trefoil domain. Additionally, this work hasdemonstrated that amino acid changes in these two β-hairpin turns canincrease the stability of the β-trefoil domain, which in turn results inincreased binding activity, see, e.g., Stephen R. Brych et al.,Structure and Stability Effects of Mutations Designed to Increase thePrimary Sequence Symmetry Within the Core Region of a β-trefoil, 10Protein Sci. 2587-2599 (2001); Jaewon Kim et al., Alternative Type I andI′ Turn Conformations in the β8/β9 β-hairpin of Human Acidic FibroblastGrowth Factor, 11 Protein Sci. 459-466 (2002); Jaewon Kim et al.,Sequence swapping Does Not Result in Conformation Swapping for the β4/β5and β8/β9 β-hairpin Turns in Human Acidic Fibroblast Growth Factor, 14Protein Sci. 351-359 (2005). As a non-limiting example, replacement ofan amino acid comprising either the β4/β5 hairpin turn or β8/β9β-hairpin turn with a glycine results in increased stabilization of theβ-trefoil domain. Therefore, replacement of amino acids located in theβ4/β5 and β8/β9 β-hairpin turns of the β-trefoil domains present in thebinding domain of Clostridial toxins will increase binding activity ofsuch a modified Clostridial toxin by increasing the structural stabilityof the β-trefoil domain. The amino acid sequences comprising theβ-trefoil domains found in various Clostridial toxins are shown in Table2.

TABLE 2 β-trefoil Domains of Clostridial Toxins Amino Acid SequenceRegion of Carbohydrate Binding Moieties β4/β5 β8/β9 Protein SEQ ID NO:α-fold β-hairpin turn β-fold β-hairpin turn γ-fold BoNT/A 1 1111-11621163-1178 1179-1223 1224-1236 1237-1296 BoNT/B 2 1098-1147 1148-11651166-1210 1211-1222 1223-1291 BoNT/C1 3 1112-1150 1151-1166 1167-12181219-1229 1230-1291 BoNT/D 4 1099-1137 1138-1153 1154-1207 1208-12181219-1276 BoNT/E 5 1086-1129 1130-1146 1147-1190 1191-1198 1199-1252BoNT/F 6 1106-1152 1153-1171 1172-1213 1214-1221 1222-1274 BoNT/G 71106-1153 1154-1172 1173-1218 1219-1230 1231-1297 TeNT 8 1128-11771178-1194 1195-1240 1241-1254 1255-1315

As is typical for proteins containing a β-trefoil fold, the overallamino acid sequence identity of the H_(CC) domain between Clostridialtoxins is low. However, key residues essential for binding activity havebeen identified by structural analysis and mutagenesis experiments, see,e.g., Krzysztof Ginalski et al., Structure-based Sequence Alignment forthe Beta-Trefoil Subdomain of the Clostridial Neurotoxin Family ProvidesResidue Level Information About the Putative Ganglioside Binding Site,482(1-2) FEBS Lett. 119-124 (2000). For example, analysis of the H_(CC)domain structure by crystallography identified five highly conservedresidues critical for forming a shallow surface pocket of a carbohydratebinding moiety. These polar residues make hydrogen bonds with thecarbohydrate ring. In BoNT/A these five polar residues are Glu 1203, Phe1252, Ser 1264, Tyr 1267 and Gly 1279, while in TeNT, these residues areAsp 1222, Thr 1270, Ser 1287, Tyr 1290 and Gly 1300. Additionally,tyrosine residues forming the hydrophilic wall of this pocket were alsoimportant (Trp 1266 of BoNT/A and Trp 1289 of TeNT) and tryptophanfluorescence quenching experiments indicated that Trp 1266 of BoNT/Abound carbohydrate molecules. In another studies, photoaffinity labelingexperiments revealed that Gln 1270 of BoNT/A and His 1293 of TeNT werealso involved in binding carbohydrate molecules. Mutagenesis experimentsdesigned to assay loss-of-function binding activity mutations confirmedthe importance of many of the residues described above for BoNT/A andTeNT and extended this analysis to BoNT/B (Glu 1190, His 1241, Typ 1262,Tyr 1263), see, e.g., Andreas Rummel et al., The H _(CC)-Domain ofBotulinum Neurotoxins A and B Exhibits a Singular Ganglioside BindingSite Displaying Serotype Specific Carbohydrate Interaction, 51(3) Mol.Microbiol. 631-643 (2004).

As used herein, the term “Clostridial toxin H_(CC) targeting domain”means any naturally occurring Clostridial toxin polypeptide that canexecute the cell binding step of the intoxication process, including,e.g., the selective binding of the Clostridial toxin to a toxin-specificreceptor located on the plasma membrane surface of a target cell. Asused herein, the term “modified Clostridial toxin H_(CC) targetingdomain” means a naturally occurring Clostridial toxin H_(CC) targetingdomain modified to enhance its cell binding activity for an endogenousClostridial toxin receptor, such as, e.g., a binding affinity or abinding specificity, to a statistically significantly degree relative tothe unmodified naturally occurring Clostridial toxin H_(CC) targetingdomain from which the modified Clostridial toxin H_(CC) targeting domainwas derived. By definition, a modified Clostridial toxin H_(CC)targeting domain has at least one amino acid change from thecorresponding region of the disclosed reference sequences (see Table 1)and can be described in percent identity to the corresponding region ofthat reference sequence.

As another non-limiting examples, a modified BoNT/A H_(CC) targetingdomain comprising amino acids 1092-1296 of SEQ ID NO: 1 will have atleast one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region1092-1296 of SEQ ID NO: 1; a modified BoNT/B H_(CC) targeting domaincomprising amino acids 1079-1291 of SEQ ID NO: 2 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1079-1291 ofSEQ ID NO: 2; a modified BoNT/C1 H_(CC) targeting domain comprisingamino acids 1093-1291 of SEQ ID NO: 3 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 2093-1291 of SEQ ID NO:3; a modified BoNT/D H_(CC) targeting domain comprising amino acids1080-1276 of SEQ ID NO: 4 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 1080-1276 of SEQ ID NO: 4; a modifiedBoNT/E H_(CC) targeting domain comprising amino acids 1067-1252 of SEQID NO: 5 will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to the aminoacid region 1067-1252 of SEQ ID NO: 5; a modified BoNT/F H_(CC)targeting domain comprising amino acids 1087-1274 of SEQ ID NO: 6 willhave at least one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to the amino acid region1087-1274 of SEQ ID NO: 6; a modified BoNT/G H_(CC) targeting domaincomprising amino acids 1087-1297 of SEQ ID NO: 7 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1087-1297 ofSEQ ID NO: 7; and a modified TeNT H_(CC) targeting domain comprisingamino acids 1109-1315 of SEQ ID NO: 8 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the amino acid region 1109-1315 of SEQ ID NO:8.

It is also envisioned that any of a variety of modified Clostridialtoxin H_(CC) targeting domain fragments comprising a binding domain withenhanced binding activity can be useful in aspects of the presentinvention with the proviso that these active fragments can provideenhanced binding activity of the toxin to the receptor located at thesurface of the target cell. The H_(CC) targeting domain from the heavychains of Clostridial toxins are approximately 165-195 amino acids inlength and comprise a binding domain (Table 1). Research has shown thatthe entire length of a H_(CC) targeting domain from a Clostridial toxinheavy chain is not necessary for the binding activity of the bindingdomain. Thus, aspects of this embodiment can include a Clostridial toxinH_(CC) targeting domain comprising a binding domain having a length of,e.g., at least 150 amino acids, at least 175 amino acids, at least 200amino acids and at least 225 amino acids. Other aspects of thisembodiment can include a Clostridial toxin H_(CC) targeting domaincomprising a binding domain having a length of, e.g., at most 150 aminoacids, at most 175 amino acids, at most 200 amino acids and at most 225amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of a modified Clostridial toxin H_(CC) targeting domainrelative to a naturally-occurring Clostridial toxin H_(CC) targetingdomain, including, without limitation, global methods, local methods andhybrid methods, such as, e.g., segment approach methods. Protocols todetermine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

One general approach well known to one skilled in the art on how tomodify a Clostridial toxin H_(CC) targeting domain in order to increaseits binding activity for a naturally-occurring Clostridial toxinreceptor present on a naturally-occurring Clostridial toxin target cellinvolves changing specifically-identified amino acids. As describedabove, amino acids essential to binding activity have been identifiedand methods useful for determining which amino acid substitutions willenhance binding activity are known to one skilled in the art. Forexample, recent advances in computational protein design algorithms havemarkedly improved capabilities for generating novel proteins withoptimized properties, including, enhanced stability, altered substratespecificity, improved binding affinity and optimized pharmacokinetics,see, e.g., Tanja Kortemme et al., Computational redesign ofprotein-protein interaction specificity, 11(4) Nat. Struct. Mol. Biol.371-379 (2004); Tanja Kortemme and David Baker, Computational design ofprotein-protein interactions, 8(1) Curr. Opin. Chem. Biol. 91-97 (2004);Motomu Shimaoka et al., Computational design of an integrin I domainstabilized in the open high affinity conformation, 7(8) Nat. Struct.Biol. 674-678 (2000); Loïc Martin et al., Rational Design of a CD4 Mimicthat Inhibits HIV-1 Entry and Exposes Cryptic Neutralization Epitopes,21(1) Nat. Biotechnol. 71-76 (2003); and Casim A. Sarkar et al.,Rational Cytokine Design for Increased Lifetime and Enhanced PotencyUsing pH-activated “Histidine Switching,”20(9) Nat. Biotechnol. 908-913(2002).

In addition, methods capable of altering an amino acid present in aClostridial toxin H_(CC) targeting domain, include, without limitation,site-directed mutagenesis, oligonucleotide-directed mutagenesis andsite-specific mutagenesis. Non-limiting examples of specific mutagenesisprotocols for making mutations in a Clostridial toxin are described in,e.g., Mutagenesis, pp. 13.1-13.105 (Sambrook and Russell, eds.,Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed. 2001). Inaddition, non-limiting examples of well-characterized mutagenesisprotocols available from commercial vendors include, without limitation,Altered Sites® II in vitro Mutagenesis Systems (Promega Corp., Madison,Wis.); Erase-a-Base® System (Promega, Madison, Wis.); GeneTailor™Site-Directed Mutagenesis System (Invitrogen, Inc., Carlsbad, Calif.);QuikChange® II Site-Directed Mutagenesis Kits (Stratagene, La Jolla,Calif.); and Transformer™ Site-Directed Mutagenesis Kit (BD-Clontech,Mountain View, Calif.).

Lastly, methods to test the binding activity of modified Clostridialtoxins comprising a modified Clostridial toxin H_(CC) targeting domainwith altered binding capability are also well known to one skilled inthe art, see, e.g., Lutea A. A. de Jong et al., Receptor-Binding Assays:Technologies and Applications, 829 J. Chromatogr. B 1-25 (2005). It isenvisioned that heterogeneous assay types, homogeneous assay types andnon-separating homogeneous assay types can be used to determine thebinding activity of a modified Clostridial toxin with enhanced bindingactivity disclosed in the present specification. In a heterogeneousassay, the free ligand is separated from the bound ligand by, e.g.,filtration, centrifugation or dialysis, before measurement of thebinding activity. In a homogeneous assay, no separation of the freeligand from the bound ligand is required before measurement of thebinding activity. In non-separating homogeneous assays, either theligand or the receptor is immobilized on a solid phase support, inaddition to the no separation aspect of all homogeneous assay.Non-limiting examples of heterogeneous, homogeneous and non-separatinghomogeneous assays include, e.g., radioactive heterogeneous assays, suchas, e.g., filtration assays using radioactive energy transfer, SPA/flashplate assays using radioactive energy transfer; and non-radioactiveheterogeneous assays, such as, e.g., filtration assays usingfluorescence, FRET assays using fluorescence energy transfer, FP assaysusing light polarization, single-cell FMAT assays and flow cytometry. Inaddition, non-limiting examples of well-characterized receptor bindingprotocols available from commercial vendors include, without limitation,Homogeneous Time Resolved Fluorescense-based receptor binding assays(HTRF®; Cisbio International, Bedford, Mass.); DELFIA®-based receptorbinding assays (PerkinElmer Lifesciences, Boston, Mass.); andAlphaScreen™-based receptor binding assays (PerkinElmer Lifesciences,Boston, Mass.).

It is further envisioned that the binding activity of a modifiedClostridial toxin with altered binding capability disclosed in thepresent specification can be determined by affinity chromatography usingimmobilized receptors and interfacial optical assays, such as, e.g.,total internal reflection fluorescence (TIRF) and surface plasmonresonance (SPR). Non-limiting examples of these assays include, e.g.,FCS using diffusion mediated intensity fluctuations, SPR using amass-dependent refractive index, TIRF using a mass-independentrefractive index, microarrays using optical intensity changes and QACusing retention volume.

As another general approach well known to one skilled in the art on howto modify a Clostridial toxin H_(CC) targeting domain in order toincrease its binding activity for a naturally-occurring Clostridialtoxin receptor present on a naturally-occurring Clostridial toxin targetcell involves directed-evolution methods, see, e.g., Andre Koltermann etal., Process for Generating Sequence-Specific Proteases by DirectedEvolution and Uses Thereof, U.S. Patent Publication 2004/0072276 (Apr.15, 2004); Lance E. Steward and Kei Roger Aoki, Evolved ClostridialToxins with Altered Protease Activity, U.S. Patent Publication2004/0115727 (Jun. 17, 2004); and L Yuan et al., Laboratory-directedprotein evolution, 69(3) Microbiol. Mol. Biol. Rev. 373-392 (2005).

Often the first step of directed evolution is error-prone PCRamplification of the gene of interest or gene recombination when afamily of related genes is available. In this case, the genecorresponding to full-length Clostridial toxin or the H_(CC) targetingdomain alone would be amplified under conditions that yield one to threeamino acid substitutions per molecule. The protein would then beexpressed and screened for the desired activity (i.e., receptor bindingand/or enhanced activity). Any constructs with even a nominalimprovement in receptor binding, regardless of the magnitude of thechange, would be submitted for additional rounds of evolution. As aresult of the random mutagenesis studies, any amino acids displayingimproved binding or even dramatically reduced binding could then besubmitted to saturation mutagenesis. This is a process in which thecodon of interest it completely randomized so that different constructscontaining each of the 20 amino acids substituted at the site ofinterest are generated.

For improvement of receptor binding characteristics, several differentscreening approaches could be utilized. If the receptor is known thesoluble portion of the receptor can be expressed recombinantly for usein SPR binding assays. For example, the soluble portion of the receptorcan be expressed as a fusion to streptavidin, polyhistidine tag, etc.and the receptor can then be immobilized on an appropriate sensor chip.Utilizing a SPR instrument, changes in local refractive index as aresult of receptor binding are measured as a change in the SPR angle.The rates of change in the SPR angle can then be analyzed to determineassociation and dissociation phases and therefore equilibrium constants.This type of assay could be measured either with the full-lengthClostridial toxin or the H_(CC) targeting domain. Additionally, bindingtype assays relying on affinity pull-down experiments with affinitytagged receptors acting as a “bait molecule” could be used. The modifiedClostridial toxin or H_(CC) targeting domain could then be labeled withradioisotopes for analysis of the amount of target protein associatedwith the bait. Alternatively, radiolabeled ligands can be competed withClostridial toxin or H_(CC) targeting domain variants that are notlabeled.

If the receptor is not known, variants of full-length Clostridial toxinscan be screened using cells that are known to be sensitive to treatmentwith native the Clostridial toxin. In this case there are severalpotential readouts for improved receptor binding, including the presenceof the Clostridial toxin in the cell, measurement of cleaved SNAREprotein by, e.g., Western blot or cell-based FRET activity assay, orinhibition of exocytosis, e.g., a neurotransmitter release assay.

Thus, in an embodiment, a modified Clostridial toxin comprising atargeting domain with enhanced binding activity comprises a modifiedClostridial toxin H_(CC) targeting domain with enhanced bindingactivity. In an aspect of this embodiment, a modified Clostridial toxinH_(CC) targeting domain comprises a modified Clostridial toxin H_(CC)targeting domain with an increased binding affinity or a modifiedClostridial toxin H_(CC) targeting domain fragment with an increasedbinding affinity. In another aspect of this embodiment, a modifiedClostridial toxin binding domain comprises a modified Clostridial toxinH_(CC) targeting domain with an increased binding specificity or amodified Clostridial toxin H_(CC) targeting domain fragment with anincreased binding specificity.

In another embodiment, a modified Clostridial toxin comprising atargeting domain with enhanced binding activity comprises a modifiedBoNT/A H_(CC) targeting domain with enhanced binding activity. In anaspect of this embodiment, a modified Clostridial toxin comprises amodified BoNT/A H_(CC) targeting domain with an increased bindingaffinity or a modified BoNT/A H_(CC) targeting domain fragment with anincreased binding affinity. In another aspect of this embodiment, amodified Clostridial toxin comprises a modified BoNT/A H_(CC) targetingdomain with an increased binding specificity or a modified BoNT/A H_(CC)targeting domain fragment with an increased binding specificity.

In another embodiment, a modified BoNT/A H_(CC) targeting domain withenhanced binding activity comprises a modification of amino acids1111-1296 of SEQ ID NO: 1. In another aspect of this embodiment, amodified BoNT/A H_(CC) targeting domain with an altered cell bindingcapability comprises a modified α-fold motif of a β-trefoil domain of aBoNT/A H_(CC) targeting domain, a modified β-fold motif of a β-trefoildomain of a BoNT/A H_(CC) targeting domain, or a modified γ-fold motifof a β-trefoil domain of a BoNT/A H_(CC) targeting domain. In anotheraspect of this embodiment, a modified BoNT/A H_(CC) targeting domainwith an altered cell binding capability comprises a modification toamino acids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296of SEQ ID NO: 1.

In other aspects of this embodiment, a modified BoNT/A H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1111-1162, amino acids 1179-1223, or amino acids1237-1296 of SEQ ID NO: 1, at least 75% amino acid identity with aminoacids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQID NO: 1, at least 80% amino acid identity with amino acids 1111-1162,amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, atleast 85% amino acid identity with amino acids 1111-1162, amino acids1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at least 90% aminoacid identity with amino acids 1111-1162, amino acids 1179-1223, oramino acids 1237-1296 of SEQ ID NO: 1 or at least 95% amino acididentity with amino acids 1111-1162, amino acids 1179-1223, or aminoacids 1237-1296 of SEQ ID NO: 1. In yet other aspects of thisembodiment, a modified BoNT/A H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQID NO: 1, at most 75% amino acid identity with amino acids 1111-1162,amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at most80% amino acid identity with amino acids 1111-1162, amino acids1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1, at most 85% aminoacid identity with amino acids 1111-1162, amino acids 1179-1223, oramino acids 1237-1296 of SEQ ID NO: 1, at most 90% amino acid identitywith amino acids 1111-1162, amino acids 1179-1223, or amino acids1237-1296 of SEQ ID NO: 1 or at most 95% amino acid identity with aminoacids 1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQID NO: 1.

In other aspects of this embodiment, a modified BoNT/A H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1111-1162, amino acids 1179-1223,or amino acids 1237-1296 of SEQ ID NO: 1. In other aspects of thisembodiment, a modified BoNT/A H_(CC) targeting domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 1111-1162, amino acids1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. In yet otheraspects of this embodiment, a modified BoNT/A H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 1111-1162, amino acids 1179-1223, oramino acids 1237-1296 of SEQ ID NO: 1. In other aspects of thisembodiment, a modified BoNT/A H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 1111-1162, amino acids 1179-1223, or amino acids1237-1296 of SEQ ID NO: 1. In still other aspects of this embodiment, amodified BoNT/A H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids1111-1162, amino acids 1179-1223, or amino acids 1237-1296 of SEQ IDNO: 1. In other aspects of this embodiment, a modified BoNT/A H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1111-1162, amino acids1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a modified BoNT/A H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1111-1162, amino acids 1179-1223,or amino acids 1237-1296 of SEQ ID NO: 1. In other aspects of thisembodiment, a modified BoNT/A H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 1111-1162, amino acids 1179-1223, or amino acids1237-1296 of SEQ ID NO: 1. In yet other aspects of this embodiment, amodified BoNT/A H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 1111-1162,amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. Inother aspects of this embodiment, a modified BoNT/A H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 1111-1162, amino acids 1179-1223, or amino acids1237-1296 of SEQ ID NO: 1. In still other aspects of this embodiment, amodified BoNT/A H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 1111-1162,amino acids 1179-1223, or amino acids 1237-1296 of SEQ ID NO: 1. Inother aspects of this embodiment, a modified BoNT/A H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1111-1162, amino acids 1179-1223, or amino acids1237-1296 of SEQ ID NO: 1.

In another embodiment, a modified BoNT/A H_(CC) targeting domain withenhanced binding activity comprises a modified BoNT/A H_(CC) targetingdomain with enhanced binding activity of comprises a modified β4/β5hairpin turn of a β-trefoil domain of a BoNT/A H_(CC) targeting domainor a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/A H_(CC)targeting domain. In another aspect of this embodiment, a modifiedBoNT/A H_(CC) targeting domain with enhanced binding activity comprisesa modification of amino acids 1163-1178 or amino acids 1224-1236 of SEQID NO: 1.

In other aspects of this embodiment, a modified BoNT/A H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, atleast 75% amino acid identity with amino acids 1163-1178 or amino acids1224-1236 of SEQ ID NO: 1, at least 80% amino acid identity with aminoacids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, at least 85%amino acid identity with amino acids 1163-1178 or amino acids 1224-1236of SEQ ID NO: 1, at least 90% amino acid identity with amino acids1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 or at least 95% aminoacid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQID NO: 1. In yet other aspects of this embodiment, a modified BoNT/AH_(CC) targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1163-1178 or amino acids 1224-1236 of SEQID NO: 1, at most 75% amino acid identity with amino acids 1163-1178 oramino acids 1224-1236 of SEQ ID NO: 1, at most 80% amino acid identitywith amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1, atmost 85% amino acid identity with amino acids 1163-1178 or amino acids1224-1236 of SEQ ID NO: 1, at most 90% amino acid identity with aminoacids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 or at most 95%amino acid identity with amino acids 1163-1178 or amino acids 1224-1236of SEQ ID NO: 1.

In other aspects of this embodiment, a modified BoNT/A H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to amino acids 1163-1178 or amino acids 1224-1236of SEQ ID NO: 1. In other aspects of this embodiment, a modified BoNT/AH_(CC) targeting domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a modified BoNT/A H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1163-1178 oramino acids 1224-1236 of SEQ ID NO: 1. In other aspects of thisembodiment, a modified BoNT/A H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In stillother aspects of this embodiment, a modified BoNT/A H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ IDNO: 1. In other aspects of this embodiment, a modified BoNT/A H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 non-contiguousamino acid additions relative to amino acids 1163-1178 or amino acids1224-1236 of SEQ ID NO: 1.

In other aspects of this embodiment, a modified BoNT/A H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid substitutionsrelative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ IDNO: 1. In other aspects of this embodiment, a modified BoNT/A H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 contiguous aminoacid substitutions relative to amino acids 1163-1178 or amino acids1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 1163-1178 oramino acids 1224-1236 of SEQ ID NO: 1 can be replaced withphenylalanine. In yet other aspects of this embodiment, a modifiedBoNT/A H_(CC) targeting domain comprising a β-trefoil fold domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid deletions relative to amino acids 1163-1178 or amino acids1224-1236 of SEQ ID NO: 1. In other aspects of this embodiment, amodified BoNT/A H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid deletions relative to amino acids 1163-1178 oramino acids 1224-1236 of SEQ ID NO: 1. In still other aspects of thisembodiment, a modified BoNT/A H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO: 1. In otheraspects of this embodiment, a modified BoNT/A H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid additionsrelative to amino acids 1163-1178 or amino acids 1224-1236 of SEQ ID NO:1.

In other aspects of this embodiment, a modified BoNT/A H_(CC) targetingdomain with enhanced binding activity comprises a substitution of aminoacid Trp 1101, Gly 1102, Leu 1105, Tyr 1111, Tyr 1112, Gly 1158, Ile1163, Asp 1179, Glu 1203, Phe 1252, Ser 1264, Trp 1266, Tyr 1267, Gln1270, Gly 1279 or Trp 1282, or any combination thereof, the substitutionenhancing the binding activity of the modified BoNT/A H_(CC) targetingdomain. In other aspects of this embodiment, a modified BoNT/A H_(CC)targeting domain with enhanced binding activity comprises a deletion ofamino acid Trp 1101, Gly 1102, Leu 1105, Tyr 1111, Tyr 1112, Gly 1158,Ile 1163, Asp 1179, Glu 1203, Phe 1252, Ser 1264, Trp 1266, Tyr 1267,Gln 1270, Gly 1279 or Trp 1282, or any combination thereof, the deletionenhancing the binding activity of the modified BoNT/A H_(CC) targetingdomain.

In another embodiment, a modified Clostridial toxin comprising atargeting domain with enhanced binding activity comprises a modifiedBoNT/B H_(CC) targeting domain with enhanced binding activity. In anaspect of this embodiment, a modified Clostridial toxin comprises amodified BoNT/B H_(CC) targeting domain with an increased bindingaffinity or a modified BoNT/B H_(CC) targeting domain fragment with anincreased binding affinity. In another aspect of this embodiment, amodified Clostridial toxin comprises a modified BoNT/B H_(CC) targetingdomain with an increased binding specificity or a modified BoNT/B H_(CC)targeting domain fragment with an increased binding specificity.

In another embodiment, a modified BoNT/B H_(CC) targeting domain withenhanced binding activity comprises a modification of amino acids1098-1291 of SEQ ID NO: 2. In another aspect of this embodiment, amodified BoNT/B H_(CC) targeting domain with an altered cell bindingcapability comprises a modified α-fold motif of a β-trefoil domain of aBoNT/B H_(CC) targeting domain, a modified β-fold motif of a β-trefoildomain of a BoNT/B H_(CC) targeting domain, or a modified γ-fold motifof a β-trefoil domain of a BoNT/B H_(CC) targeting domain. In anotheraspect of this embodiment, a modified BoNT/B H_(CC) targeting domainwith an altered cell binding capability comprises a modification toamino acids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291of SEQ ID NO: 2.

In other aspects of this embodiment, a modified BoNT/B H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1098-1147, amino acids 1166-1210, or amino acids1223-1291 of SEQ ID NO: 2, at least 75% amino acid identity with aminoacids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQID NO: 2, at least 80% amino acid identity with amino acids 1098-1147,amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, atleast 85% amino acid identity with amino acids 1098-1147, amino acids1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at least 90% aminoacid identity with amino acids 1098-1147, amino acids 1166-1210, oramino acids 1223-1291 of SEQ ID NO: 2 or at least 95% amino acididentity with amino acids 1098-1147, amino acids 1166-1210, or aminoacids 1223-1291 of SEQ ID NO: 2. In yet other aspects of thisembodiment, a modified BoNT/B H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQID NO: 2, at most 75% amino acid identity with amino acids 1098-1147,amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at most80% amino acid identity with amino acids 1098-1147, amino acids1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2, at most 85% aminoacid identity with amino acids 1098-1147, amino acids 1166-1210, oramino acids 1223-1291 of SEQ ID NO: 2, at most 90% amino acid identitywith amino acids 1098-1147, amino acids 1166-1210, or amino acids1223-1291 of SEQ ID NO: 2 or at most 95% amino acid identity with aminoacids 1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQID NO: 2.

In other aspects of this embodiment, a modified BoNT/B H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1098-1147, amino acids 1166-1210,or amino acids 1223-1291 of SEQ ID NO: 2. In other aspects of thisembodiment, a modified BoNT/B H_(CC) targeting domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 1098-1147, amino acids1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. In yet otheraspects of this embodiment, a modified BoNT/B H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 1098-1147, amino acids 1166-1210, oramino acids 1223-1291 of SEQ ID NO: 2. In other aspects of thisembodiment, a modified BoNT/B H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 1098-1147, amino acids 1166-1210, or amino acids1223-1291 of SEQ ID NO: 2. In still other aspects of this embodiment, amodified BoNT/B H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids1098-1147, amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO:2. In other aspects of this embodiment, a modified BoNT/B H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1098-1147, amino acids1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2.

In other aspects of this embodiment, a modified BoNT/B H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1098-1147, amino acids 1166-1210,or amino acids 1223-1291 of SEQ ID NO: 2. In other aspects of thisembodiment, a modified BoNT/B H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 1098-1147, amino acids 1166-1210, or amino acids1223-1291 of SEQ ID NO: 2. In yet other aspects of this embodiment, amodified BoNT/B H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 1098-1147,amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. Inother aspects of this embodiment, a modified BoNT/B H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 1098-1147, amino acids 1166-1210, or amino acids1223-1291 of SEQ ID NO: 2. In still other aspects of this embodiment, amodified BoNT/B H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 1098-1147,amino acids 1166-1210, or amino acids 1223-1291 of SEQ ID NO: 2. Inother aspects of this embodiment, a modified BoNT/B H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1098-1147, amino acids 1166-1210, or amino acids1223-1291 of SEQ ID NO: 2.

In another embodiment, a modified BoNT/B H_(CC) targeting domain withenhanced binding activity comprises a modified BoNT/B H_(CC) targetingdomain with enhanced binding activity of comprises a modified β4/β5hairpin turn of a β-trefoil domain of a BoNT/B H_(CC) targeting domainor a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/B H_(CC)targeting domain. In another aspect of this embodiment, a modifiedBoNT/B H_(CC) targeting domain with enhanced binding activity comprisesa modification of amino acids 1148-1165 or amino acids 1211-1222 of SEQID NO: 2.

In other aspects of this embodiment, a modified BoNT/B H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, atleast 75% amino acid identity with amino acids 1148-1165 or amino acids1211-1222 of SEQ ID NO: 2, at least 80% amino acid identity with aminoacids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, at least 85%amino acid identity with amino acids 1148-1165 or amino acids 1211-1222of SEQ ID NO: 2, at least 90% amino acid identity with amino acids1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 or at least 95% aminoacid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQID NO: 2. In yet other aspects of this embodiment, a modified BoNT/BH_(CC) targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1148-1165 or amino acids 1211-1222 of SEQID NO: 2, at most 75% amino acid identity with amino acids 1148-1165 oramino acids 1211-1222 of SEQ ID NO: 2, at most 80% amino acid identitywith amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2, atmost 85% amino acid identity with amino acids 1148-1165 or amino acids1211-1222 of SEQ ID NO: 2, at most 90% amino acid identity with aminoacids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 or at most 95%amino acid identity with amino acids 1148-1165 or amino acids 1211-1222of SEQ ID NO: 2.

In other aspects of this embodiment, a modified BoNT/B H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to amino acids 1148-1165 or amino acids 1211-1222of SEQ ID NO: 2. In other aspects of this embodiment, a modified BoNT/BH_(CC) targeting domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a modified BoNT/B H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1148-1165 oramino acids 1211-1222 of SEQ ID NO: 2. In other aspects of thisembodiment, a modified BoNT/B H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In stillother aspects of this embodiment, a modified BoNT/B H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO:2. In other aspects of this embodiment, a modified BoNT/B H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 non-contiguousamino acid additions relative to amino acids 1148-1165 or amino acids1211-1222 of SEQ ID NO: 2.

In other aspects of this embodiment, a modified BoNT/B H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid substitutionsrelative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO:2. In other aspects of this embodiment, a modified BoNT/B H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 contiguous aminoacid substitutions relative to amino acids 1148-1165 or amino acids1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 1148-1165 oramino acids 1211-1222 of SEQ ID NO: 2 can be replaced withphenylalanine. In yet other aspects of this embodiment, a modifiedBoNT/B H_(CC) targeting domain comprising a β-trefoil fold domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid deletions relative to amino acids 1148-1165 or amino acids1211-1222 of SEQ ID NO: 2. In other aspects of this embodiment, amodified BoNT/B H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid deletions relative to amino acids 1148-1165 oramino acids 1211-1222 of SEQ ID NO: 2. In still other aspects of thisembodiment, a modified BoNT/B H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO: 2. In otheraspects of this embodiment, a modified BoNT/B H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid additionsrelative to amino acids 1148-1165 or amino acids 1211-1222 of SEQ ID NO:2.

In other aspects of this embodiment, a modified BoNT/B H_(CC) targetingdomain with enhanced binding activity comprises a substitution of aminoacid Trp 1088, Gly 1089, Leu 1092, Tyr 1098, Tyr 1099, Gly 1142, Ile1147, Asp 1165, Glu 1191, Ile 1240, Ser 1260, Trp 1262, Tyr 1263, Glu1266, Gly 1277 or Trp 1280, or any combination thereof, the substitutionenhancing the binding capability of the modified BoNT/B H_(CC) targetingdomain. In other aspects of this embodiment, a modified BoNT/B H_(CC)targeting domain with enhanced binding activity comprises a deletion ofamino acid Trp 1088, Gly 1089, Leu 1092, Tyr 1098, Tyr 1099, Gly 1142,Ile 1147, Asp 1165, Glu 1191, Ile 1240, Ser 1260, Trp 1262, Tyr 1263,Glu 1266, Gly 1277 or Trp 1280, or any combination thereof, the deletionenhancing the binding capability of the modified BoNT/B H_(CC) targetingdomain.

In another embodiment, a modified Clostridial toxin comprising atargeting domain with enhanced binding activity comprises a modifiedBoNT/C1 H_(CC) targeting domain with enhanced binding activity. In anaspect of this embodiment, a modified Clostridial toxin comprises amodified BoNT/C1 H_(CC) targeting domain with an increased bindingaffinity or a modified BoNT/C1 H_(CC) targeting domain fragment with anincreased binding affinity. In another aspect of this embodiment, amodified Clostridial toxin comprises a modified BoNT/C1 H_(CC) targetingdomain with an increased binding specificity or a modified BoNT/C1H_(CC) targeting domain fragment with an increased binding specificity.

In another embodiment, a modified BoNT/C1 H_(CC) targeting domain withenhanced binding activity comprises a modification of amino acids1112-1291 of SEQ ID NO: 3. In another aspect of this embodiment, amodified BoNT/C1 H_(CC) targeting domain with an altered cell bindingcapability comprises a modified α-fold motif of a β-trefoil domain of aBoNT/C1 H_(CC) targeting domain, a modified β-fold motif of a β-trefoildomain of a BoNT/C1 H_(CC) targeting domain, or a modified γ-fold motifof a β-trefoil domain of a BoNT/C1 H_(CC) targeting domain. In anotheraspect of this embodiment, a modified BoNT/C1 H_(CC) targeting domainwith an altered cell binding capability comprises a modification toamino acids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291of SEQ ID NO: 3.

In other aspects of this embodiment, a modified BoNT/C1 H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1112-1150, amino acids 1167-1218, or amino acids1230-1291 of SEQ ID NO: 3, at least 75% amino acid identity with aminoacids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQID NO: 3, at least 80% amino acid identity with amino acids 1112-1150,amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, atleast 85% amino acid identity with amino acids 1112-1150, amino acids1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at least 90% aminoacid identity with amino acids 1112-1150, amino acids 1167-1218, oramino acids 1230-1291 of SEQ ID NO: 3 or at least 95% amino acididentity with amino acids 1112-1150, amino acids 1167-1218, or aminoacids 1230-1291 of SEQ ID NO: 3. In yet other aspects of thisembodiment, a modified BoNT/C1 H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQID NO: 3, at most 75% amino acid identity with amino acids 1112-1150,amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at most80% amino acid identity with amino acids 1112-1150, amino acids1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3, at most 85% aminoacid identity with amino acids 1112-1150, amino acids 1167-1218, oramino acids 1230-1291 of SEQ ID NO: 3, at most 90% amino acid identitywith amino acids 1112-1150, amino acids 1167-1218, or amino acids1230-1291 of SEQ ID NO: 3 or at most 95% amino acid identity with aminoacids 1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQID NO: 3.

In other aspects of this embodiment, a modified BoNT/C1 H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1112-1150, amino acids 1167-1218,or amino acids 1230-1291 of SEQ ID NO: 3. In other aspects of thisembodiment, a modified BoNT/C1 H_(CC) targeting domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 1112-1150, amino acids1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. In yet otheraspects of this embodiment, a modified BoNT/C1 H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 1112-1150, amino acids 1167-1218, oramino acids 1230-1291 of SEQ ID NO: 3. In other aspects of thisembodiment, a modified BoNT/C1 H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 1112-1150, amino acids 1167-1218, or amino acids1230-1291 of SEQ ID NO: 3. In still other aspects of this embodiment, amodified BoNT/C1 H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids1112-1150, amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO:3. In other aspects of this embodiment, a modified BoNT/C1 H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1112-1150, amino acids1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3.

In other aspects of this embodiment, a modified BoNT/C1 H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1112-1150, amino acids 1167-1218,or amino acids 1230-1291 of SEQ ID NO: 3. In other aspects of thisembodiment, a modified BoNT/C1 H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 1112-1150, amino acids 1167-1218, or amino acids1230-1291 of SEQ ID NO: 3. In yet other aspects of this embodiment, amodified BoNT/C1 H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 1112-1150,amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. Inother aspects of this embodiment, a modified BoNT/C1 H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 1112-1150, amino acids 1167-1218, or amino acids1230-1291 of SEQ ID NO: 3. In still other aspects of this embodiment, amodified BoNT/C1 H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 1112-1150,amino acids 1167-1218, or amino acids 1230-1291 of SEQ ID NO: 3. Inother aspects of this embodiment, a modified BoNT/C1 H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1112-1150, amino acids 1167-1218, or amino acids1230-1291 of SEQ ID NO: 3.

In another embodiment, a modified BoNT/C1 H_(CC) targeting domain withenhanced binding activity comprises a modified BoNT/C1 H_(CC) targetingdomain with enhanced binding activity of comprises a modified β4/β5hairpin turn of a β-trefoil domain of a BoNT/C1 H_(CC) targeting domainor a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/C1 H_(CC)targeting domain. In another aspect of this embodiment, a modifiedBoNT/C1 H_(CC) targeting domain with enhanced binding activity comprisesa modification of amino acids 1151-1166 or amino acids 1219-1229 of SEQID NO: 3.

In other aspects of this embodiment, a modified BoNT/C1 H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, atleast 75% amino acid identity with amino acids 1151-1166 or amino acids1219-1229 of SEQ ID NO: 3, at least 80% amino acid identity with aminoacids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, at least 85%amino acid identity with amino acids 1151-1166 or amino acids 1219-1229of SEQ ID NO: 3, at least 90% amino acid identity with amino acids1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 or at least 95% aminoacid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQID NO: 3. In yet other aspects of this embodiment, a modified BoNT/C1H_(CC) targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1151-1166 or amino acids 1219-1229 of SEQID NO: 3, at most 75% amino acid identity with amino acids 1151-1166 oramino acids 1219-1229 of SEQ ID NO: 3, at most 80% amino acid identitywith amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3, atmost 85% amino acid identity with amino acids 1151-1166 or amino acids1219-1229 of SEQ ID NO: 3, at most 90% amino acid identity with aminoacids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 or at most 95%amino acid identity with amino acids 1151-1166 or amino acids 1219-1229of SEQ ID NO: 3.

In other aspects of this embodiment, a modified BoNT/C1 H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to amino acids 1151-1166 or amino acids 1219-1229of SEQ ID NO: 3. In other aspects of this embodiment, a modified BoNT/C1H_(CC) targeting domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a modified BoNT/C1 H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1151-1166 oramino acids 1219-1229 of SEQ ID NO: 3. In other aspects of thisembodiment, a modified BoNT/C1 H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In stillother aspects of this embodiment, a modified BoNT/C1 H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO:3. In other aspects of this embodiment, a modified BoNT/C1 H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 non-contiguousamino acid additions relative to amino acids 1151-1166 or amino acids1219-1229 of SEQ ID NO: 3.

In other aspects of this embodiment, a modified BoNT/C1 H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid substitutionsrelative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO:3. In other aspects of this embodiment, a modified BoNT/C1 H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 contiguous aminoacid substitutions relative to amino acids 1151-1166 or amino acids1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 1151-1166 oramino acids 1219-1229 of SEQ ID NO: 3 can be replaced withphenylalanine. In yet other aspects of this embodiment, a modifiedBoNT/C1 H_(CC) targeting domain comprising a β-trefoil fold domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid deletions relative to amino acids 1151-1166 or amino acids1219-1229 of SEQ ID NO: 3. In other aspects of this embodiment, amodified BoNT/C1 H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid deletions relative to amino acids 1151-1166 oramino acids 1219-1229 of SEQ ID NO: 3. In still other aspects of thisembodiment, a modified BoNT/C1 H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO: 3. In otheraspects of this embodiment, a modified BoNT/C1 H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid additionsrelative to amino acids 1151-1166 or amino acids 1219-1229 of SEQ ID NO:3.

In other aspects of this embodiment, a modified BoNT/C1 H_(CC) targetingdomain with enhanced binding activity comprises a substitution of aminoacid Trp 1102, Gly 1103, Leu 1106, Tyr 1112, Tyr 1113, Gly 1145, Ile1150, Asp 1166, Glu 1196, Ile 1247, Gly 1256, Trp 1258, Tyr 1259, His1261, Gly 1281 or Trp 1284, or any combination thereof, the substitutionenhancing the binding activity of the modified BoNT/C1 H_(CC) targetingdomain. In other aspects of this embodiment, a modified BoNT/C1 H_(CC)targeting domain with enhanced binding activity comprises a deletion ofamino acid Trp 1102, Gly 1103, Leu 1106, Tyr 1112, Tyr 1113, Gly 1145,Ile 1150, Asp 1166, Glu 1196, Ile 1247, Gly 1256, Trp 1258, Tyr 1259,His 1261, Gly 1281 or Trp 1284, or any combination thereof, the deletionenhancing the binding activity of the modified BoNT/C1 H_(CC) targetingdomain.

In another embodiment, a modified Clostridial toxin comprising atargeting domain with enhanced binding activity comprises a modifiedBoNT/D H_(CC) targeting domain with enhanced binding activity. In anaspect of this embodiment, a modified Clostridial toxin comprises amodified BoNT/D H_(CC) targeting domain with an increased bindingaffinity or a modified BoNT/D H_(CC) targeting domain fragment with anincreased binding affinity. In another aspect of this embodiment, amodified Clostridial toxin comprises a modified BoNT/D H_(CC) targetingdomain with an increased binding specificity or a modified BoNT/D H_(CC)targeting domain fragment with an increased binding specificity.

In another embodiment, a modified BoNT/D H_(CC) targeting domain withenhanced binding activity comprises a modification of amino acids1099-1276 of SEQ ID NO: 4. In another aspect of this embodiment, amodified BoNT/D H_(CC) targeting domain with an altered cell bindingcapability comprises a modified α-fold motif of a β-trefoil domain of aBoNT/D H_(CC) targeting domain, a modified β-fold motif of a β-trefoildomain of a BoNT/D H_(CC) targeting domain, or a modified γ-fold motifof a β-trefoil domain of a BoNT/D H_(CC) targeting domain. In anotheraspect of this embodiment, a modified BoNT/D H_(CC) targeting domainwith an altered cell binding capability comprises a modification toamino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276of SEQ ID NO: 4.

In other aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least 70% amino acididentity with amino acids 1099-1137, amino acids 1154-1207, or aminoacids 1219-1276 of SEQ ID NO: 4, at least 75% amino acid identity withamino acids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276of SEQ ID NO: 4, at least 80% amino acid identity with amino acids1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO:4, at least 85% amino acid identity with amino acids 1099-1137, aminoacids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at least 90%amino acid identity with amino acids 1099-1137, amino acids 1154-1207,or amino acids 1219-1276 of SEQ ID NO: 4 or at least 95% amino acididentity with amino acids 1099-1137, amino acids 1154-1207, or aminoacids 1219-1276 of SEQ ID NO: 4. In yet other aspects of thisembodiment, a modified BoNT/D H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQID NO: 4, at most 75% amino acid identity with amino acids 1099-1137,amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at most80% amino acid identity with amino acids 1099-1137, amino acids1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4, at most 85% aminoacid identity with amino acids 1099-1137, amino acids 1154-1207, oramino acids 1219-1276 of SEQ ID NO: 4, at most 90% amino acid identitywith amino acids 1099-1137, amino acids 1154-1207, or amino acids1219-1276 of SEQ ID NO: 4 or at most 95% amino acid identity with aminoacids 1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQID NO: 4.

In other aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1099-1137, amino acids 1154-1207,or amino acids 1219-1276 of SEQ ID NO: 4. In other aspects of thisembodiment, a modified BoNT/D H_(CC) targeting domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 1099-1137, amino acids1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. In yet otheraspects of this embodiment, a modified BoNT/D H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 1099-1137, amino acids 1154-1207, oramino acids 1219-1276 of SEQ ID NO: 4. In other aspects of thisembodiment, a modified BoNT/D H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 1099-1137, amino acids 1154-1207, or amino acids1219-1276 of SEQ ID NO: 4. In still other aspects of this embodiment, amodified BoNT/D H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids1099-1137, amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO:4. In other aspects of this embodiment, a modified BoNT/D H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1099-1137, amino acids1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4.

In other aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1099-1137, amino acids 1154-1207,or amino acids 1219-1276 of SEQ ID NO: 4. In other aspects of thisembodiment, a modified BoNT/D H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 1099-1137, amino acids 1154-1207, or amino acids1219-1276 of SEQ ID NO: 4. In yet other aspects of this embodiment, amodified BoNT/D H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 1099-1137,amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. Inother aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 1099-1137, amino acids 1154-1207, or amino acids1219-1276 of SEQ ID NO: 4. In still other aspects of this embodiment, amodified BoNT/D H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 1099-1137,amino acids 1154-1207, or amino acids 1219-1276 of SEQ ID NO: 4. Inother aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1099-1137, amino acids 1154-1207, or amino acids1219-1276 of SEQ ID NO: 4.

In another embodiment, a modified BoNT/D H_(CC) targeting domain withenhanced binding activity comprises a modified BoNT/D H_(CC) targetingdomain with enhanced binding activity of comprises a modified β4/β5hairpin turn of a β-trefoil domain of a BoNT/D H_(CC) targeting domainor a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/D H_(CC)targeting domain. In another aspect of this embodiment, a modifiedBoNT/D H_(CC) targeting domain with enhanced binding activity comprisesa modification of amino acids 1138-1153 or amino acids 1208-1218 of SEQID NO: 4.

In other aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least 70% amino acididentity with amino acids 1138-1153 or amino acids 1208-1218 of SEQ IDNO: 4, at least 75% amino acid identity with amino acids 1138-1153 oramino acids 1208-1218 of SEQ ID NO: 4, at least 80% amino acid identitywith amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, atleast 85% amino acid identity with amino acids 1138-1153 or amino acids1208-1218 of SEQ ID NO: 4, at least 90% amino acid identity with aminoacids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 or at least 95%amino acid identity with amino acids 1138-1153 or amino acids 1208-1218of SEQ ID NO: 4. In yet other aspects of this embodiment, a modifiedBoNT/D H_(CC) targeting domain comprising a β-trefoil fold domain withenhanced binding activity comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1138-1153 or amino acids1208-1218 of SEQ ID NO: 4, at most 75% amino acid identity with aminoacids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at most 80%amino acid identity with amino acids 1138-1153 or amino acids 1208-1218of SEQ ID NO: 4, at most 85% amino acid identity with amino acids1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4, at most 90% aminoacid identity with amino acids 1138-1153 or amino acids 1208-1218 of SEQID NO: 4 or at most 95% amino acid identity with amino acids 1138-1153or amino acids 1208-1218 of SEQ ID NO: 4.

In other aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to amino acids 1138-1153 or amino acids 1208-1218of SEQ ID NO: 4. In other aspects of this embodiment, a modified BoNT/DH_(CC) targeting domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a modified BoNT/D H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1138-1153 oramino acids 1208-1218 of SEQ ID NO: 4. In other aspects of thisembodiment, a modified BoNT/D H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In stillother aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO:4. In other aspects of this embodiment, a modified BoNT/D H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 non-contiguousamino acid additions relative to amino acids 1138-1153 or amino acids1208-1218 of SEQ ID NO: 4.

In other aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 contiguous amino acidsubstitutions relative to amino acids 1138-1153 or amino acids 1208-1218of SEQ ID NO: 4. In other aspects of this embodiment, a modified BoNT/DH_(CC) targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 contiguous aminoacid substitutions relative to amino acids 1138-1153 or amino acids1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 1138-1153 oramino acids 1208-1218 of SEQ ID NO: 4 can be replaced withphenylalanine. In yet other aspects of this embodiment, a modifiedBoNT/D H_(CC) targeting domain comprising a β-trefoil fold domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid deletions relative to amino acids 1138-1153 or amino acids1208-1218 of SEQ ID NO: 4. In other aspects of this embodiment, amodified BoNT/D H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid deletions relative to amino acids 1138-1153 oramino acids 1208-1218 of SEQ ID NO: 4. In still other aspects of thisembodiment, a modified BoNT/D H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO: 4. In otheraspects of this embodiment, a modified BoNT/D H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid additionsrelative to amino acids 1138-1153 or amino acids 1208-1218 of SEQ ID NO:4.

In other aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain with enhanced binding activity comprises a substitution of aminoacid Trp 1089, Gly 1090, Leu 1093, Tyr 1099, Tyr 1100, Gly 1132, Ile1137, Asp 1153, Asn 1186, Lys 1236, Trp 1238, Arg 1239, Phe 1242, Ser1262 or Trp 1265, or any combination thereof, the substitution enhancingthe binding activity of the modified BoNT/D H_(CC) targeting domain. Inother aspects of this embodiment, a modified BoNT/D H_(CC) targetingdomain with enhanced binding activity comprises a deletion of amino acidTrp 1089, Gly 1090, Leu 1093, Tyr 1099, Tyr 1100, Gly 1132, Ile 1137,Asp 1153, Asn 1186, Lys 1236, Trp 1238, Arg 1239, Phe 1242, Ser 1262 orTrp 1265, or any combination thereof, the deletion enhancing the bindingactivity of the modified BoNT/D H_(CC) targeting domain.

In another embodiment, a modified Clostridial toxin comprising atargeting domain with enhanced binding activity comprises a modifiedBoNT/E H_(CC) targeting domain with enhanced binding activity. In anaspect of this embodiment, a modified Clostridial toxin comprises amodified BoNT/E H_(CC) targeting domain with an increased bindingaffinity or a modified BoNT/E H_(CC) targeting domain fragment with anincreased binding affinity. In another aspect of this embodiment, amodified Clostridial toxin comprises a modified BoNT/E H_(CC) targetingdomain with an increased binding specificity or a modified BoNT/E H_(CC)targeting domain fragment with an increased binding specificity.

In another embodiment, a modified BoNT/E H_(CC) targeting domain withenhanced binding activity comprises a modification of amino acids1086-1252 of SEQ ID NO: 5. In another aspect of this embodiment, amodified BoNT/E H_(CC) targeting domain with an altered cell bindingcapability comprises a modified α-fold motif of a β-trefoil domain of aBoNT/E H_(CC) targeting domain, a modified β-fold motif of a β-trefoildomain of a BoNT/E H_(CC) targeting domain, or a modified γ-fold motifof a β-trefoil domain of a BoNT/E H_(CC) targeting domain. In anotheraspect of this embodiment, a modified BoNT/E H_(CC) targeting domainwith an altered cell binding capability comprises a modification toamino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252of SEQ ID NO: 5.

In other aspects of this embodiment, a modified BoNT/E H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least 70% amino acididentity with amino acids 1086-1129, amino acids 1147-1190, or aminoacids 1199-1252 of SEQ ID NO: 5, at least 75% amino acid identity withamino acids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252of SEQ ID NO: 5, at least 80% amino acid identity with amino acids1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO:5, at least 85% amino acid identity with amino acids 1086-1129, aminoacids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at least 90%amino acid identity with amino acids 1086-1129, amino acids 1147-1190,or amino acids 1199-1252 of SEQ ID NO: 5 or at least 95% amino acididentity with amino acids 1086-1129, amino acids 1147-1190, or aminoacids 1199-1252 of SEQ ID NO: 5. In yet other aspects of thisembodiment, a modified BoNT/E H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQID NO: 5, at most 75% amino acid identity with amino acids 1086-1129,amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at most80% amino acid identity with amino acids 1086-1129, amino acids1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5, at most 85% aminoacid identity with amino acids 1086-1129, amino acids 1147-1190, oramino acids 1199-1252 of SEQ ID NO: 5, at most 90% amino acid identitywith amino acids 1086-1129, amino acids 1147-1190, or amino acids1199-1252 of SEQ ID NO: 5 or at most 95% amino acid identity with aminoacids 1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQID NO: 5.

In other aspects of this embodiment, a modified BoNT/E H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1086-1129, amino acids 1147-1190,or amino acids 1199-1252 of SEQ ID NO: 5. In other aspects of thisembodiment, a modified BoNT/E H_(CC) targeting domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 1086-1129, amino acids1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. In yet otheraspects of this embodiment, a modified BoNT/E H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 1086-1129, amino acids 1147-1190, oramino acids 1199-1252 of SEQ ID NO: 5. In other aspects of thisembodiment, a modified BoNT/E H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 1086-1129, amino acids 1147-1190, or amino acids1199-1252 of SEQ ID NO: 5. In still other aspects of this embodiment, amodified BoNT/E H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids1086-1129, amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO:5. In other aspects of this embodiment, a modified BoNT/E H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1086-1129, amino acids1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5.

In other aspects of this embodiment, a modified BoNT/E H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1086-1129, amino acids 1147-1190,or amino acids 1199-1252 of SEQ ID NO: 5. In other aspects of thisembodiment, a modified BoNT/E H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 1086-1129, amino acids 1147-1190, or amino acids1199-1252 of SEQ ID NO: 5. In yet other aspects of this embodiment, amodified BoNT/E H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 1086-1129,amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. Inother aspects of this embodiment, a modified BoNT/E H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 1086-1129, amino acids 1147-1190, or amino acids1199-1252 of SEQ ID NO: 5. In still other aspects of this embodiment, amodified BoNT/E H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 1086-1129,amino acids 1147-1190, or amino acids 1199-1252 of SEQ ID NO: 5. Inother aspects of this embodiment, a modified BoNT/E H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1086-1129, amino acids 1147-1190, or amino acids1199-1252 of SEQ ID NO: 5.

In another embodiment, a modified BoNT/E H_(CC) targeting domain withenhanced binding activity comprises a modified BoNT/E H_(CC) targetingdomain with enhanced binding activity of comprises a modified β4/β5hairpin turn of a β-trefoil domain of a BoNT/E H_(CC) targeting domainor a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/E H_(CC)targeting domain. In another aspect of this embodiment, a modifiedBoNT/E H_(CC) targeting domain with enhanced binding activity comprisesa modification of amino acids 1130-1146 or amino acids 1191-1198 of SEQID NO: 5.

In other aspects of this embodiment, a modified BoNT/E H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least 70% amino acididentity with amino acids 1130-1146 or amino acids 1191-1198 of SEQ IDNO: 5, at least 75% amino acid identity with amino acids 1130-1146 oramino acids 1191-1198 of SEQ ID NO: 5, at least 80% amino acid identitywith amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, atleast 85% amino acid identity with amino acids 1130-1146 or amino acids1191-1198 of SEQ ID NO: 5, at least 90% amino acid identity with aminoacids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 or at least 95%amino acid identity with amino acids 1130-1146 or amino acids 1191-1198of SEQ ID NO: 5. In yet other aspects of this embodiment, a modifiedBoNT/E H_(CC) targeting domain comprising a β-trefoil fold domain withenhanced binding activity comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1130-1146 or amino acids1191-1198 of SEQ ID NO: 5, at most 75% amino acid identity with aminoacids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at most 80%amino acid identity with amino acids 1130-1146 or amino acids 1191-1198of SEQ ID NO: 5, at most 85% amino acid identity with amino acids1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5, at most 90% aminoacid identity with amino acids 1130-1146 or amino acids 1191-1198 of SEQID NO: 5 or at most 95% amino acid identity with amino acids 1130-1146or amino acids 1191-1198 of SEQ ID NO: 5.

In other aspects of this embodiment, a modified BoNT/E H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to amino acids 1130-1146 or amino acids 1191-1198of SEQ ID NO: 5. In other aspects of this embodiment, a modified BoNT/EH_(CC) targeting domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a modified BoNT/E H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1130-1146 oramino acids 1191-1198 of SEQ ID NO: 5. In other aspects of thisembodiment, a modified BoNT/E H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In stillother aspects of this embodiment, a modified BoNT/E H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO:5. In other aspects of this embodiment, a modified BoNT/E H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 non-contiguousamino acid additions relative to amino acids 1130-1146 or amino acids1191-1198 of SEQ ID NO: 5.

In other aspects of this embodiment, a modified BoNT/E H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid substitutionsrelative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO:5. In other aspects of this embodiment, a modified BoNT/E H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 contiguous aminoacid substitutions relative to amino acids 1130-1146 or amino acids1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 1130-1146 oramino acids 1191-1198 of SEQ ID NO: 5 can be replaced withphenylalanine. In yet other aspects of this embodiment, a modifiedBoNT/E H_(CC) targeting domain comprising a β-trefoil fold domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid deletions relative to amino acids 1130-1146 or amino acids1191-1198 of SEQ ID NO: 5. In other aspects of this embodiment, amodified BoNT/E H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid deletions relative to amino acids 1130-1146 oramino acids 1191-1198 of SEQ ID NO: 5. In still other aspects of thisembodiment, a modified BoNT/E H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO: 5. In otheraspects of this embodiment, a modified BoNT/E H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid additionsrelative to amino acids 1130-1146 or amino acids 1191-1198 of SEQ ID NO:5.

In other aspects of this embodiment, a modified BoNT/E H_(CC) targetingdomain with enhanced binding activity comprises a substitution of aminoacid Trp 1076, Gly 1077, Leu 1080, Tyr 1086, Tyr 1087, Gly 1124, Ile1129, Asp 1146, Glu 1172, Phe 1213, Ser 1221, Trp 1223, Tyr 1224, His1227, Gly 1236 or Trp 1239, or any combination thereof, the substitutionenhancing the binding activity of the modified BoNT/E H_(CC) targetingdomain. In other aspects of this embodiment, a modified BoNT/E H_(CC)targeting domain with enhanced binding activity comprises a deletion ofamino acid Trp 1076, Gly 1077, Leu 1080, Tyr 1086, Tyr 1087, Gly 1124,Ile 1129, Asp 1146, Glu 1172, Phe 1213, Ser 1221, Trp 1223, Tyr 1224,His 1227, Gly 1236 or Trp 1239, or any combination thereof, the deletionenhancing the binding activity of the modified BoNT/E H_(CC) targetingdomain.

In another embodiment, a modified Clostridial toxin comprising atargeting domain with enhanced binding activity comprises a modifiedBoNT/F H_(CC) targeting domain with enhanced binding activity. In anaspect of this embodiment, a modified Clostridial toxin comprises amodified BoNT/F H_(CC) targeting domain with an increased bindingaffinity or a modified BoNT/F H_(CC) targeting domain fragment with anincreased binding affinity. In another aspect of this embodiment, amodified Clostridial toxin comprises a modified BoNT/F H_(CC) targetingdomain with an increased binding specificity or a modified BoNT/F H_(CC)targeting domain fragment with an increased binding specificity.

In another embodiment, a modified BoNT/F H_(CC) targeting domain withenhanced binding activity comprises a modification of amino acids1106-1274 of SEQ ID NO: 6. In another aspect of this embodiment, amodified BoNT/F H_(CC) targeting domain with an altered cell bindingcapability comprises a modified α-fold motif of a β-trefoil domain of aBoNT/F H_(CC) targeting domain, a modified β-fold motif of a β-trefoildomain of a BoNT/F H_(CC) targeting domain, or a modified γ-fold motifof a β-trefoil domain of a BoNT/F H_(CC) targeting domain. In anotheraspect of this embodiment, a modified BoNT/F H_(CC) targeting domainwith an altered cell binding capability comprises a modification toamino acids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274of SEQ ID NO: 6.

In other aspects of this embodiment, a modified BoNT/F H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1106-1152, amino acids 1172-1213, or amino acids1222-1274 of SEQ ID NO: 6, at least 75% amino acid identity with aminoacids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQID NO: 6, at least 80% amino acid identity with amino acids 1106-1152,amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, atleast 85% amino acid identity with amino acids 1106-1152, amino acids1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at least 90% aminoacid identity with amino acids 1106-1152, amino acids 1172-1213, oramino acids 1222-1274 of SEQ ID NO: 6 or at least 95% amino acididentity with amino acids 1106-1152, amino acids 1172-1213, or aminoacids 1222-1274 of SEQ ID NO: 6. In yet other aspects of thisembodiment, a modified BoNT/F H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQID NO: 6, at most 75% amino acid identity with amino acids 1106-1152,amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at most80% amino acid identity with amino acids 1106-1152, amino acids1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6, at most 85% aminoacid identity with amino acids 1106-1152, amino acids 1172-1213, oramino acids 1222-1274 of SEQ ID NO: 6, at most 90% amino acid identitywith amino acids 1106-1152, amino acids 1172-1213, or amino acids1222-1274 of SEQ ID NO: 6 or at most 95% amino acid identity with aminoacids 1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQID NO: 6.

In other aspects of this embodiment, a modified BoNT/F H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1106-1152, amino acids 1172-1213,or amino acids 1222-1274 of SEQ ID NO: 6. In other aspects of thisembodiment, a modified BoNT/F H_(CC) targeting domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 1106-1152, amino acids1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. In yet otheraspects of this embodiment, a modified BoNT/F H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 1106-1152, amino acids 1172-1213, oramino acids 1222-1274 of SEQ ID NO: 6. In other aspects of thisembodiment, a modified BoNT/F H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 1106-1152, amino acids 1172-1213, or amino acids1222-1274 of SEQ ID NO: 6. In still other aspects of this embodiment, amodified BoNT/F H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids1106-1152, amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO:6. In other aspects of this embodiment, a modified BoNT/F H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1106-1152, amino acids1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6.

In other aspects of this embodiment, a modified BoNT/F H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1106-1152, amino acids 1172-1213,or amino acids 1222-1274 of SEQ ID NO: 6. In other aspects of thisembodiment, a modified BoNT/F H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 1106-1152, amino acids 1172-1213, or amino acids1222-1274 of SEQ ID NO: 6. In yet other aspects of this embodiment, amodified BoNT/F H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 1106-1152,amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. Inother aspects of this embodiment, a modified BoNT/F H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 1106-1152, amino acids 1172-1213, or amino acids1222-1274 of SEQ ID NO: 6. In still other aspects of this embodiment, amodified BoNT/F H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 1106-1152,amino acids 1172-1213, or amino acids 1222-1274 of SEQ ID NO: 6. Inother aspects of this embodiment, a modified BoNT/F H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1106-1152, amino acids 1172-1213, or amino acids1222-1274 of SEQ ID NO: 6.

In another embodiment, a modified BoNT/F H_(CC) targeting domain withenhanced binding activity comprises a modified BoNT/F H_(CC) targetingdomain with enhanced binding activity of comprises a modified β4/β5hairpin turn of a β-trefoil domain of a BoNT/F H_(CC) targeting domainor a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/F H_(CC)targeting domain. In another aspect of this embodiment, a modifiedBoNT/F H_(CC) targeting domain with enhanced binding activity comprisesa modification of amino acids 1153-1171 or amino acids 1214-1221 of SEQID NO: 6.

In other aspects of this embodiment, a modified BoNT/F H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, atleast 75% amino acid identity with amino acids 1153-1171 or amino acids1214-1221 of SEQ ID NO: 6, at least 80% amino acid identity with aminoacids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, at least 85%amino acid identity with amino acids 1153-1171 or amino acids 1214-1221of SEQ ID NO: 6, at least 90% amino acid identity with amino acids1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 or at least 95% aminoacid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQID NO: 6. In yet other aspects of this embodiment, a modified BoNT/FH_(CC) targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1153-1171 or amino acids 1214-1221 of SEQID NO: 6, at most 75% amino acid identity with amino acids 1153-1171 oramino acids 1214-1221 of SEQ ID NO: 6, at most 80% amino acid identitywith amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6, atmost 85% amino acid identity with amino acids 1153-1171 or amino acids1214-1221 of SEQ ID NO: 6, at most 90% amino acid identity with aminoacids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 or at most 95%amino acid identity with amino acids 1153-1171 or amino acids 1214-1221of SEQ ID NO: 6.

In other aspects of this embodiment, a modified BoNT/F H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to amino acids 1153-1171 or amino acids 1214-1221of SEQ ID NO: 6. In other aspects of this embodiment, a modified BoNT/FH_(CC) targeting domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a modified BoNT/F H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1153-1171 oramino acids 1214-1221 of SEQ ID NO: 6. In other aspects of thisembodiment, a modified BoNT/F H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In stillother aspects of this embodiment, a modified BoNT/F H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO:6. In other aspects of this embodiment, a modified BoNT/F H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 non-contiguousamino acid additions relative to amino acids 1153-1171 or amino acids1214-1221 of SEQ ID NO: 6.

In other aspects of this embodiment, a modified BoNT/F H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid substitutionsrelative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO:6. In other aspects of this embodiment, a modified BoNT/F H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 contiguous aminoacid substitutions relative to amino acids 1153-1171 or amino acids1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 1153-1171 oramino acids 1214-1221 of SEQ ID NO: 6 can be replaced withphenylalanine. In yet other aspects of this embodiment, a modifiedBoNT/F H_(CC) targeting domain comprising a β-trefoil fold domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid deletions relative to amino acids 1153-1171 or amino acids1214-1221 of SEQ ID NO: 6. In other aspects of this embodiment, amodified BoNT/F H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid deletions relative to amino acids 1153-1171 oramino acids 1214-1221 of SEQ ID NO: 6. In still other aspects of thisembodiment, a modified BoNT/F H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO: 6. In otheraspects of this embodiment, a modified BoNT/F H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid additionsrelative to amino acids 1153-1171 or amino acids 1214-1221 of SEQ ID NO:6.

In other aspects of this embodiment, a modified BoNT/F H_(CC) targetingdomain with enhanced binding activity comprises a substitution of aminoacid Trp 1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1147, Ile1152, Asp 1171, Glu 1195, Phe 1237, Ser 1245, Trp 1247, Tyr 1248, Asn1251, Gly 1260 or Trp 1263, or any combination thereof, the substitutionenhancing the binding activity of the modified BoNT/F H_(CC) targetingdomain. In other aspects of this embodiment, a modified BoNT/F H_(CC)targeting domain with enhanced binding activity comprises a deletion ofamino acid Trp 1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1147,Ile 1152, Asp 1171, Glu 1195, Phe 1237, Ser 1245, Trp 1247, Tyr 1248,Asn 1251, Gly 1260 or Trp 1263, or any combination thereof, the deletionenhancing the binding activity of the modified BoNT/F H_(CC) targetingdomain.

In another embodiment, a modified Clostridial toxin comprising atargeting domain with enhanced binding activity comprises a modifiedBoNT/G H_(CC) targeting domain with enhanced binding activity. In anaspect of this embodiment, a modified Clostridial toxin comprises amodified BoNT/G H_(CC) targeting domain with an increased bindingaffinity or a modified BoNT/G H_(CC) targeting domain fragment with anincreased binding affinity. In another aspect of this embodiment, amodified Clostridial toxin comprises a modified BoNT/G H_(CC) targetingdomain with an increased binding specificity or a modified BoNT/G H_(CC)targeting domain fragment with an increased binding specificity.

In another embodiment, a modified BoNT/G H_(CC) targeting domain withenhanced binding activity comprises a modification of amino acids1106-1297 of SEQ ID NO: 7. In another aspect of this embodiment, amodified BoNT/G H_(CC) targeting domain with an altered cell bindingcapability comprises a modified α-fold motif of a β-trefoil domain of aBoNT/G H_(CC) targeting domain, a modified β-fold motif of a β-trefoildomain of a BoNT/G H_(CC) targeting domain, or a modified γ-fold motifof a β-trefoil domain of a BoNT/G H_(CC) targeting domain. In anotheraspect of this embodiment, a modified BoNT/G H_(CC) targeting domainwith an altered cell binding capability comprises a modification toamino acids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297of SEQ ID NO: 7.

In other aspects of this embodiment, a modified BoNT/G H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1106-1153, amino acids 1173-1218, or amino acids1231-1297 of SEQ ID NO: 7, at least 75% amino acid identity with aminoacids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQID NO: 7, at least 80% amino acid identity with amino acids 1106-1153,amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, atleast 85% amino acid identity with amino acids 1106-1153, amino acids1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at least 90% aminoacid identity with amino acids 1106-1153, amino acids 1173-1218, oramino acids 1231-1297 of SEQ ID NO: 7 or at least 95% amino acididentity with amino acids 1106-1153, amino acids 1173-1218, or aminoacids 1231-1297 of SEQ ID NO: 7. In yet other aspects of thisembodiment, a modified BoNT/G H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQID NO: 7, at most 75% amino acid identity with amino acids 1106-1153,amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at most80% amino acid identity with amino acids 1106-1153, amino acids1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7, at most 85% aminoacid identity with amino acids 1106-1153, amino acids 1173-1218, oramino acids 1231-1297 of SEQ ID NO: 7, at most 90% amino acid identitywith amino acids 1106-1153, amino acids 1173-1218, or amino acids1231-1297 of SEQ ID NO: 7 or at most 95% amino acid identity with aminoacids 1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQID NO: 7.

In other aspects of this embodiment, a modified BoNT/G H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1106-1153, amino acids 1173-1218,or amino acids 1231-1297 of SEQ ID NO: 7. In other aspects of thisembodiment, a modified BoNT/G H_(CC) targeting domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 1106-1153, amino acids1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. In yet otheraspects of this embodiment, a modified BoNT/G H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 1106-1153, amino acids 1173-1218, oramino acids 1231-1297 of SEQ ID NO: 7. In other aspects of thisembodiment, a modified BoNT/G H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 1106-1153, amino acids 1173-1218, or amino acids1231-1297 of SEQ ID NO: 7. In still other aspects of this embodiment, amodified BoNT/G H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid additions relative to amino acids1106-1153, amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO:7. In other aspects of this embodiment, a modified BoNT/G H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1106-1153, amino acids1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7.

In other aspects of this embodiment, a modified BoNT/G H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1106-1153, amino acids 1173-1218,or amino acids 1231-1297 of SEQ ID NO: 7. In other aspects of thisembodiment, a modified BoNT/G H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 1106-1153, amino acids 1173-1218, or amino acids1231-1297 of SEQ ID NO: 7. In yet other aspects of this embodiment, amodified BoNT/G H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid deletions relative to amino acids 1106-1153,amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. Inother aspects of this embodiment, a modified BoNT/G H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 1106-1153, amino acids 1173-1218, or amino acids1231-1297 of SEQ ID NO: 7. In still other aspects of this embodiment, amodified BoNT/G H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid additions relative to amino acids 1106-1153,amino acids 1173-1218, or amino acids 1231-1297 of SEQ ID NO: 7. Inother aspects of this embodiment, a modified BoNT/G H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1106-1153, amino acids 1173-1218, or amino acids1231-1297 of SEQ ID NO: 7.

In another embodiment, a modified BoNT/G H_(CC) targeting domain withenhanced binding activity comprises a modified BoNT/G H_(CC) targetingdomain with enhanced binding activity of comprises a modified β4/β5hairpin turn of a β-trefoil domain of a BoNT/G H_(CC) targeting domainor a β8/β9 hairpin turn of a β-trefoil domain of a BoNT/G H_(CC)targeting domain. In another aspect of this embodiment, a modifiedBoNT/G H_(CC) targeting domain with enhanced binding activity comprisesa modification of amino acids 1154-1172 or amino acids 1219-1230 of SEQID NO: 7.

In other aspects of this embodiment, a modified BoNT/G H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, atleast 75% amino acid identity with amino acids 1154-1172 or amino acids1219-1230 of SEQ ID NO: 7, at least 80% amino acid identity with aminoacids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, at least 85%amino acid identity with amino acids 1154-1172 or amino acids 1219-1230of SEQ ID NO: 7, at least 90% amino acid identity with amino acids1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 or at least 95% aminoacid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQID NO: 7. In yet other aspects of this embodiment, a modified BoNT/GH_(CC) targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1154-1172 or amino acids 1219-1230 of SEQID NO: 7, at most 75% amino acid identity with amino acids 1154-1172 oramino acids 1219-1230 of SEQ ID NO: 7, at most 80% amino acid identitywith amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7, atmost 85% amino acid identity with amino acids 1154-1172 or amino acids1219-1230 of SEQ ID NO: 7, at most 90% amino acid identity with aminoacids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 or at most 95%amino acid identity with amino acids 1154-1172 or amino acids 1219-1230of SEQ ID NO: 7.

In other aspects of this embodiment, a modified BoNT/G H_(CC) targetingdomain comprising a 11-trefoil fold domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to amino acids 1154-1172 or amino acids 1219-1230of SEQ ID NO: 7. In other aspects of this embodiment, a modified BoNT/GH_(CC) targeting domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a modified BoNT/G H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1154-1172 oramino acids 1219-1230 of SEQ ID NO: 7. In other aspects of thisembodiment, a modified BoNT/G H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In stillother aspects of this embodiment, a modified BoNT/G H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO:7. In other aspects of this embodiment, a modified BoNT/G H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 non-contiguousamino acid additions relative to amino acids 1154-1172 or amino acids1219-1230 of SEQ ID NO: 7.

In other aspects of this embodiment, a modified BoNT/G H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid substitutionsrelative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO:7. In other aspects of this embodiment, a modified BoNT/G H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 contiguous aminoacid substitutions relative to amino acids 1154-1172 or amino acids1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 1154-1172 oramino acids 1219-1230 of SEQ ID NO: 7 can be replaced withphenylalanine. In yet other aspects of this embodiment, a modifiedBoNT/G H_(CC) targeting domain comprising a β-trefoil fold domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine or 10 contiguousamino acid deletions relative to amino acids 1154-1172 or amino acids1219-1230 of SEQ ID NO: 7. In other aspects of this embodiment, amodified BoNT/G H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine or10 contiguous amino acid deletions relative to amino acids 1154-1172 oramino acids 1219-1230 of SEQ ID NO: 7. In still other aspects of thisembodiment, a modified BoNT/G H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid additions relative toamino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO: 7. In otheraspects of this embodiment, a modified BoNT/G H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid additionsrelative to amino acids 1154-1172 or amino acids 1219-1230 of SEQ ID NO:7.

In other aspects of this embodiment, a modified BoNT/G H_(CC) targetingdomain with enhanced binding activity comprises a substitution of aminoacid Trp 1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1148, Ile1153, Asp 1172, Gln 1198, Ile 1245, Ser 1266, Trp 1268, Tyr 1269, Arg1272, Gly 1283 or Trp 1285, or any combination thereof, the substitutionenhancing the binding activity of the modified BoNT/G H_(CC) targetingdomain. In other aspects of this embodiment, a modified BoNT/G H_(CC)targeting domain with enhanced binding activity comprises a deletion ofamino acid Trp 1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1148,Ile 1153, Asp 1172, Gln 1198, Ile 1245, Ser 1266, Trp 1268, Tyr 1269,Arg 1272, Gly 1283 or Trp 1285, or any combination thereof, the deletionenhancing the binding activity of the modified BoNT/G H_(CC) targetingdomain.

In another embodiment, a modified Clostridial toxin comprising atargeting domain with enhanced binding activity comprises a modifiedTeNT H_(CC) targeting domain with enhanced binding activity. In anaspect of this embodiment, a modified Clostridial toxin comprises amodified TeNT H_(CC) targeting domain with an increased binding affinityor a modified TeNT H_(CC) targeting domain fragment with an increasedbinding affinity. In another aspect of this embodiment, a modifiedClostridial toxin comprises a modified TeNT H_(CC) targeting domain withan increased binding specificity or a modified TeNT H_(CC) targetingdomain fragment with an increased binding specificity.

In another embodiment, a modified TeNT H_(CC) targeting domain withenhanced binding activity comprises a modification of amino acids1128-1315 of SEQ ID NO: 8. In another aspect of this embodiment, amodified TeNT H_(CC) targeting domain with an altered cell bindingcapability comprises a modified α-fold motif of a β-trefoil domain of aTeNT H_(CC) targeting domain, a modified β-fold motif of a β-trefoildomain of a TeNT H_(CC) targeting domain, or a modified γ-fold motif ofa β-trefoil domain of a TeNT H_(CC) targeting domain. In another aspectof this embodiment, a modified TeNT H_(CC) targeting domain with analtered cell binding capability comprises a modification to amino acids1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO:8.

In other aspects of this embodiment, a modified TeNT H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1128-1177, amino acids 1195-1240, or amino acids1255-1315 of SEQ ID NO: 8, at least 75% amino acid identity with aminoacids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQID NO: 8, at least 80% amino acid identity with amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, atleast 85% amino acid identity with amino acids 1128-1177, amino acids1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at least 90% aminoacid identity with amino acids 1128-1177, amino acids 1195-1240, oramino acids 1255-1315 of SEQ ID NO: 8 or at least 95% amino acididentity with amino acids 1128-1177, amino acids 1195-1240, or aminoacids 1255-1315 of SEQ ID NO: 8. In yet other aspects of thisembodiment, a modified TeNT H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQID NO: 8, at most 75% amino acid identity with amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at most80% amino acid identity with amino acids 1128-1177, amino acids1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8, at most 85% aminoacid identity with amino acids 1128-1177, amino acids 1195-1240, oramino acids 1255-1315 of SEQ ID NO: 8, at most 90% amino acid identitywith amino acids 1128-1177, amino acids 1195-1240, or amino acids1255-1315 of SEQ ID NO: 8 or at most 95% amino acid identity with aminoacids 1128-1177, amino acids 1195-1240, or amino acids 1255-1315 of SEQID NO: 8.

In other aspects of this embodiment, a modified TeNT H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1128-1177, amino acids 1195-1240,or amino acids 1255-1315 of SEQ ID NO: 8. In other aspects of thisembodiment, a modified TeNT H_(CC) targeting domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid substitutions relative to amino acids 1128-1177, amino acids1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In yet otheraspects of this embodiment, a modified TeNT H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 1128-1177, amino acids 1195-1240, oramino acids 1255-1315 of SEQ ID NO: 8. In other aspects of thisembodiment, a modified TeNT H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 1128-1177, amino acids 1195-1240, or amino acids1255-1315 of SEQ ID NO: 8. In still other aspects of this embodiment, amodified TeNT H_(CC) targeting domain comprising a β-trefoil fold domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1128-1177,amino acids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. Inother aspects of this embodiment, a modified TeNT H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 1128-1177, amino acids 1195-1240, oramino acids 1255-1315 of SEQ ID NO: 8.

In other aspects of this embodiment, a modified TeNT H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1128-1177, amino acids 1195-1240,or amino acids 1255-1315 of SEQ ID NO: 8. In other aspects of thisembodiment, a modified TeNT H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid substitutionsrelative to amino acids 1128-1177, amino acids 1195-1240, or amino acids1255-1315 of SEQ ID NO: 8. In yet other aspects of this embodiment, amodified TeNT H_(CC) targeting domain comprising a β-trefoil fold domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 1128-1177, aminoacids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In otheraspects of this embodiment, a modified TeNT H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 1128-1177, amino acids 1195-1240, or amino acids1255-1315 of SEQ ID NO: 8. In still other aspects of this embodiment, amodified TeNT H_(CC) targeting domain comprising a β-trefoil fold domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 1128-1177, aminoacids 1195-1240, or amino acids 1255-1315 of SEQ ID NO: 8. In otheraspects of this embodiment, a modified TeNT H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 1128-1177, amino acids 1195-1240, or amino acids1255-1315 of SEQ ID NO: 8.

In another embodiment, a modified TeNT H_(CC) targeting domain withenhanced binding activity comprises a modified TeNT H_(CC) targetingdomain with enhanced binding activity of comprises a modified β4/β5hairpin turn of a β-trefoil domain of a TeNT H_(CC) targeting domain ora β8/β9 hairpin turn of a β-trefoil domain of a TeNT H_(CC) targetingdomain. In another aspect of this embodiment, a modified TeNT H_(CC)targeting domain with enhanced binding activity comprises a modificationof amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8.

In other aspects of this embodiment, a modified TeNT H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, atleast 75% amino acid identity with amino acids 1178-1194 or amino acids1241-1254 of SEQ ID NO: 8, at least 80% amino acid identity with aminoacids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, at least 85%amino acid identity with amino acids 1178-1194 or amino acids 1241-1254of SEQ ID NO: 8, at least 90% amino acid identity with amino acids1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 or at least 95% aminoacid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQID NO: 8. In yet other aspects of this embodiment, a modified TeNTH_(CC) targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 1178-1194 or amino acids 1241-1254 of SEQID NO: 8, at most 75% amino acid identity with amino acids 1178-1194 oramino acids 1241-1254 of SEQ ID NO: 8, at most 80% amino acid identitywith amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8, atmost 85% amino acid identity with amino acids 1178-1194 or amino acids1241-1254 of SEQ ID NO: 8, at most 90% amino acid identity with aminoacids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 or at most 95%amino acid identity with amino acids 1178-1194 or amino acids 1241-1254of SEQ ID NO: 8.

In other aspects of this embodiment, a modified TeNT H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acidsubstitutions relative to amino acids 1178-1194 or amino acids 1241-1254of SEQ ID NO: 8. In other aspects of this embodiment, a modified TeNTH_(CC) targeting domain comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid substitutions relative to amino acids1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a modified TeNT H_(CC) targeting domain comprising a β-trefoil folddomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine or 10non-contiguous amino acid deletions relative to amino acids 1178-1194 oramino acids 1241-1254 of SEQ ID NO: 8. In other aspects of thisembodiment, a modified TeNT H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or 10 non-contiguous amino acid deletions relative toamino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In stillother aspects of this embodiment, a modified TeNT H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO:8. In other aspects of this embodiment, a modified TeNT H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 non-contiguous amino acid additionsrelative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO:8.

In other aspects of this embodiment, a modified TeNT H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid substitutionsrelative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO:8. In other aspects of this embodiment, a modified TeNT H_(CC) targetingdomain comprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid substitutionsrelative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO:8. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 1178-1194 or amino acids1241-1254 of SEQ ID NO: 8 can be replaced with glycine. In other aspectsof this embodiment, contiguous amino acid substitutions of hydrophobicamino acids from amino acids 1178-1194 or amino acids 1241-1254 of SEQID NO: 8 can be replaced with phenylalanine. In yet other aspects ofthis embodiment, a modified TeNT H_(CC) targeting domain comprising aβ-trefoil fold domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine or 10 contiguous amino acid deletions relative toamino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO: 8. In otheraspects of this embodiment, a modified TeNT H_(CC) targeting domaincomprising a β-trefoil fold domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or 10 contiguous amino acid deletionsrelative to amino acids 1178-1194 or amino acids 1241-1254 of SEQ ID NO:8. In still other aspects of this embodiment, a modified TeNT H_(CC)targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine or 10 contiguous amino acidadditions relative to amino acids 1178-1194 or amino acids 1241-1254 ofSEQ ID NO: 8. In other aspects of this embodiment, a modified TeNTH_(CC) targeting domain comprising a β-trefoil fold domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine or 10 contiguous aminoacid additions relative to amino acids 1178-1194 or amino acids1241-1254 of SEQ ID NO: 8.

In other aspects of this embodiment, a modified TeNT H_(CC) targetingdomain with enhanced binding activity comprises a substitution of aminoacid Trp 1118, Gly 1119, Leu 1122, Tyr 1128, Tyr 1129, Gly 1172, Ile1177, Asp 1194, Asp 1222, Thr 1270, Ser 1287, Trp 1289, Tyr 1290, His1293, Gly 1300 or Trp 1303, or any combination thereof, the substitutionenhancing the binding activity of the modified TeNT H_(CC) targetingdomain. In other aspects of this embodiment, a modified TeNT H_(CC)targeting domain with enhanced binding activity comprises a deletion ofamino acid Trp 1118, Gly 1119, Leu 1122, Tyr 1128, Tyr 1129, Gly 1172,Ile 1177, Asp 1194, Asp 1222, Thr 1270, Ser 1287, Trp 1289, Tyr 1290,His 1293, Gly 1300 or Trp 1303, or any combination thereof, the deletionenhancing the binding activity of the modified TeNT H_(CC) targetingdomain.

Another example of an enhanced targeting domain that increases bindingactivity for an endogenous Clostridial toxin receptor present on anaturally-occurring Clostridial toxin target cell, includes, withoutlimitation, non-toxin associated proteins of Clostridial toxins, suchas, e.g., non-toxic non-hemagglutinin (NTNH), hemagglutinin-17 (HA-17),hemagglutinin-33 (HA-33) and hemagglutinin-70 (HA-70). In vivo,Clostridial bacteria produce a toxin complex comprising theapproximately 150-kDa Clostridial toxin and other proteins collectivelycalled nontoxin associated proteins (NAPs). Identified NAPs includeproteins possessing hemaglutination activity, such, e.g., ahemagglutinin of approximately 17-kDa (HA-17), a hemagglutinin ofapproximately 33-kDa (HA-33) and a hemagglutinin of approximately 70-kDa(HA-70); as well as non-toxic non-hemagglutinin (NTNH), a protein ofapproximately 130-kDa, see, e.g., Eric A. Johnson and Marite Bradshaw,Clostridial botulinum and its Neurotoxins: A Metabolic and CellularPerspective, 39 Toxicon 1703-1722 (2001); and Stephanie Raffestin etal., Organization and Regulation of the Neurotoxin Genes in Clostridiumbotulinum and Clostridium tetani, 10 Anaerobe 93-100 (2004). The toxincomplex is important for the intoxication process because it providesprotection from adverse environmental conditions, resistance to proteasedigestion, and appears to facilitate internalization and activation ofthe toxin.

Recent crystallography experiments have revealed that HA-17, HA-33 andNTNH from various Clostridial bacteria contain a region comprisingβ-trefoil domains very similar to the single β-trefoil domain present inthe binding domain of Clostridial toxins, see, e.g., Kaoru Inoue et al.,Structural Analysis by X-Ray Crystallography and Calorimetry of aHaemagglutinin Component (HA1) of the Progenitor Toxin from Clostridiumbotulinum, 149 Microbiol. 3361-3370 (2003); and Joseph W. Arndt et al.,The Structure of the Neurotoxin-Associated Protein HA33/A fromClostridial botulinum Suggests a Reoccurring β-trefoil Fold in theProgenitor Toxin Complex, 346 J. Mol. Biol. 1083-1093 (2005). Forexample, HA-33 from Clostridium botulinum serotype A has two β-trefoildomains, each of which consists of three potential carbohydrate bindingmoieties or β-trefoil folds, designated 1α (amino acids 10-55 of SEQ IDNO: 9), 1β (amino acids 56-102 of SEQ ID NO: 9) and 1γ (amino acids103-144 of SEQ ID NO: 9) for the first β-trefoil domain and 2α (aminoacids 151-197 of SEQ ID NO: 9), 2β (amino acids 198-245 of SEQ ID NO: 9)and 2γ (amino acids 246-293 of SEQ ID NO: 9) for the second β-trefoildomain. Mutations in conserved amino acids of the carbohydrate bindingmoiety result in a loss of carbohydrate binding, see, e.g., Kaoru Inoueet al., Structural Analysis by X-Ray Crystallography and Calorimetry ofa Haemagglutinin Component (HA1) of the Progenitor Toxin fromClostridium botulinum, 149 Microbiol. 3361-3370 (2003).

These β-trefoil domains are also found in the HA-33 proteins produced byClostridium botulinum serotype B, serotype C1 and serotype D andsequence alignments revealed that amino acids essential for overallcarbohydrate binding are conserved. The amino acids predicted to beessential for carbohydrate binding are as follows Asp 263, Tyr 265, Gln268, Gln 276, Phe 278 and Gln 286 of HA-33/A1 of SEQ ID NO: 9, HA-33/A2of SEQ ID NO: 10, HA-33/A3 of SEQ ID NO: 11, HA-33/A5 of SEQ ID NO: 12and HA-33/B2 of SEQ ID NO: 15; Asp 264, Tyr 266, Gln 269, Gln 277, Phe279 and Gln 287 of HA-33/A4 of SEQ ID NO: 12; Asp 262, Tyr 264, Gln 267,Gln 275, Phe 277 and Gln 285 of HA-33/B1 of SEQ ID NO: 14; Asp 255, Val257, Gly 260, Gln 268, Typ 270 and Gln 278 of HA-33/C1 of SEQ ID NO: 16;and Asp 256, Tyr 258, Gln 261, Ile 269, Asp 271 and Gln 279 of HA-33/C2of SEQ ID NO: 17 and HA-33/D of SEQ ID NO: 18. Immunoaffinity columnchromatography and pull-down assays have shown that HA-33 can bindsynaptotagmin II, a putative Clostridial toxin receptor, see, e.g., YuZhou et al., Haemagglutinin-33 of Type Q Botulinum Neurotoxin ComplexBinds with Synaptotagmin II, 272 FEBS Lett. 2717-2726 (2005). The aminoacid sequences comprising the β-trefoil domains found in variousClostridial HA-33 proteins are shown in Tables 3 and 4.

TABLE 3 β-trefoil Domains of Clostridial HA-33 Proteins Amino AcidSequence Region of Carbohydrate Binding Moieties 1β4/β5 1β8/β9 ProteinSEQ ID NO: 1α-fold β-hairpin turn 1β-fold β-hairpin turn 1γ-foldHA-33/A1 9 10-54 55-59 60-100 101-104 105-144 HA-33/A2 10 10-54 55-5960-100 101-104 105-144 HA-33/A3 11 10-54 55-59 60-100 101-104 105-144HA-33/A4 12 10-56 57-61 62-102 103-106 107-146 HA-33/A5 13 10-54 55-5960-100 101-104 105-144 HA-33/B1 14 10-54 55-59 60-100 101-104 105-144HA-33/B2 15 10-56 57-61 62-102 103-106 107-146 HA-33/C1-1 16 10-54 55-5960-98   99-102 103-141 HA-33/C1-2 17 10-54 55-59 60-98   99-102 103-141HA-33/D1 18 10-54 55-59 60-98   99-102 103-141

TABLE 4 β-trefoil Domains of Clostridial HA-33 Proteins Amino AcidSequence Region of Carbohydrate Binding Moieties 2β4/β5 2β8/β9 ProteinSEQ ID NO: 2α-fold β-hairpin turn 2β-fold β-hairpin turn 2γ-foldHA-33/A1 9 151-195 196-199 200-242 243-248 249-293 HA-33/A2 10 151-195196-199 200-242 243-248 249-293 HA-33/A3 11 151-195 196-199 200-242243-248 249-293 HA-33/A4 12 153-197 198-201 202-243 244-249 250-294HA-33/A5 13 151-195 196-199 200-242 243-248 249-279 HA-33/B1 14 151-195196-199 200-241 242-247 248-292 HA-33/B2 15 153-197 198-201 200-242243-248 249-291 HA-33/C1-1 16 148-190 191-194 195-234 235-240 241-285HA-33/C1-2 17 148-190 191-194 195-235 236-241 242-286 HA-33/D1 18148-190 191-194 195-235 236-241 242-286

Further analysis of the β-trefoil domain sequence of HA-33 alsoidentified β-trefoil domains in HA-17 and NTNH, see, e.g., Joseph W.Arndt et al., The Structure of the Neurotoxin-Associated Protein HA33/Afrom Clostridial botulinum Suggests a Reoccurring β-trefoil Fold in theProgenitor Toxin Complex, 346J. Mol. Biol. 1083-1093 (2005). The HA-17comprises a single β-trefoil domain containing three carbohydratebinding moieties or β-trefoil folds. The carbohydrate binding moietiesof HA-17 exhibits the greatest sequence similarity with the 2γcarbohydrate binding moiety of HA-33. These β-trefoil domains are alsofound in the HA-17 proteins produced by Clostridium botulinum serotypeB, serotype C1 and serotype D and sequence alignments revealed thatamino acids essential for overall carbohydrate binding are conserved.The amino acids predicted to be essential for carbohydrate binding areas follows Tyr 110, Typ 112, Tyr 115, Pro 130, Phe 132 and Asn 138 ofHA-17/A of SEQ ID NO: 19, HA-17/B of SEQ ID NO: 20, HA-17/C1 of SEQ IDNO: 21 and HA-17/D of SEQ ID NO: 22. The amino acid sequences comprisingthe β-trefoil domains found in various Clostridial HA-17 proteins areshown in Table 5.

TABLE 5 β-trefoil Domains of Clostridial HA-17 Proteins Amino AcidSequence Region of Carbohydrate Binding Moieties β4/β5 β8/β9 Protein SEQID NO: α-fold β-hairpin turn β-fold β-hairpin turn γ-fold HA-17/A 199-50 51-54 55-91 92-94 95-146 HA-17/B 20 9-50 51-54 55-91 92-94 95-146HA-17/C1 21 9-50 51-54 55-91 92-94 95-146 HA-17/D 22 9-50 51-54 55-9192-94 95-146

NTNH from various Clostridial bacteria shows significant sequencesimilarity to the β-trefoil domains present in the cell binding domainof BoNT/A and TeNT. The high degree of structural similarity isinteresting in light of the low sequence similarity between NTNH and theClostridial toxins. Furthermore, since NTNH of the various serotypeshave greater sequence similarity than the Clostridial toxins, it islikely that the NTNH produced by other Clostridial strains will alsohave β-trefoil domains exhibiting high structural similarity with thebinding domains of Clostridial toxins. The β-trefoil domains of variousClostridial NTNHs are as follows: amino acids 1050-1193 of NTNH/A1 ofSEQ ID NO: 23; amino acids 1050-1198 of NTNH/A2 of SEQ ID NO: 24; aminoacids 1050-1193 of NTNH/A3 of SEQ ID NO: 25; amino acids 1049-1197 ofNTNH/B of SEQ ID NO: 26; amino acids 1049-1196 of NTNH/C1 of SEQ ID NO:27; amino acids 1049-1196 of NTNH/D of SEQ ID NO: 28; amino acids1014-1162 of NTNH/E of SEQ ID NO: 29; amino acids 1016-1159 of NTNH/F1of SEQ ID NO: 30; amino acids 1017-1165 of NTNH/F2 of SEQ ID NO: 31; andamino acids 1050-1198 of NTNH/G of SEQ ID NO: 32. The amino acidsequences comprising the β-trefoil domains found in various ClostridialNTNH proteins are shown in Table 6.

The β-trefoil domains present in the Clostridial toxin, HA-33, HA-17 andNTNH collectively form nearly half the mass of a Clostridial toxincomplex and underlies the apparent importance of carbohydrate binding inthe cell binding step of the intoxication process. This observation isfurther enhanced by the fact that a Clostridial toxin alone is not aseffective in intoxicating a cell as the entire toxin complex. Onepotential explanation for this enhanced binding activity is thepresence, both in type and in quantity, of the β-trefoil domains presentin HA-33, HA-17 and NTNH. Therefore, the high prediction of structuralsimilarity of the β-trefoil domains present in HA-33, HA-17 and NTNHrelative to the β-trefoil domain found in Clostridial toxins provides apotential source of binding domains useful for developing modifiedClostridial toxins with enhanced binding activity. As a non-limitingexample, a carbohydrate binding moiety or a β-trefoil fold from HA-33,HA-17 or NTNH can be substituted for the naturally occurringcarbohydrate binding moiety present in a Clostridial toxin. As anothernon-limiting example, a carbohydrate binding moiety or a β-trefoil foldfrom HA-33, HA-17 or NTNH can be added in addition to the naturallyoccurring carbohydrate binding moiety present in a Clostridial toxin. Asyet another non-limiting example, a multiple carbohydrate bindingmoieties or a β-trefoil folds from HA-33, HA-17 or NTNH can besubstituted for the naturally occurring carbohydrate binding moietypresent in a Clostridial toxin. As still another non-limiting example,multiple carbohydrate binding moieties or a β-trefoil folds from HA-33,HA-17 or NTNH can be added in addition to the naturally occurringcarbohydrate binding moiety present in a Clostridial toxin. As anothernon-limiting example, multiple carbohydrate binding moieties or aβ-trefoil folds from a Clostridial toxin binding domain can be added inaddition to the naturally occurring carbohydrate binding moiety presentin a Clostridial toxin.

TABLE 6 β-trefoil Domains of Clostridial NTNH Proteins Amino AcidSequence Region of Carbohydrate Binding Moieties β4/β5 β8/β9 Protein SEQID NO: α-fold β-hairpin turn β-fold β-hairpin turn γ-fold NTNH/A1 231050-1097 1098-1110 1111-1138 1139-1148 1149-1194 NTNH/A2 24 1050-10971098-1110 1111-1139 1140-1148 1149-1199 NTNH/A3 25 1050-1097 1098-11101111-1138 1139-1148 1149-1194 NTNH/B 26 1049-1096 1097-1109 1110-11381139-1147 1148-1198 NTNH/C1 27 1049-1096 1097-1109 1110-1138 1139-11471148-1197 NTNH/D 28 1049-1096 1097-1109 1110-1138 1139-1147 1148-1197NTNH/E 29 1014-1061 1062-1074 1075-1103 1104-1113 1114-1163 NTNH/F1 301016-1063 1064-1076 1077-1104 1105-1114 1115-1160 NTNH/F2 31 1017-10641065-1077 1078-1106 1107-1116 1117-1166 NTNH/G 32 1050-1097 1098-11101111-1139 1140-1149 1150-1199

As used herein, the term “Non-toxin Associated Protein” is synonymouswith “NAP” and means a Clostridial NAP with selective binding activity,such as, e.g., a binding affinity or a binding specificity, for anendogenous Clostridial toxin receptor. It is envisioned that bothnaturally occurring NAPs as well as NAPs with enhanced binding activitycan be used to practice aspects of the present invention. As usedherein, the term “NAP with enhanced binding activity” means aClostridial NAP with enhanced binding activity for an endogenousClostridial toxin receptor, such as, e.g., a binding affinity or abinding specificity, to a statistically significantly degree relative toan unmodified naturally occurring Clostridial toxin binding domain froma Clostridial toxin. By definition, a NAP with enhanced binding activityhas at least one amino acid change from the corresponding region of thedisclosed reference sequences (see Table 3-6) and can be described inpercent identity to the corresponding region of that reference sequence.

Any of a variety of sequence alignment methods can be used to determinepercent identity of a modified Clostridial NAP relative to anaturally-occurring Clostridial NAP, including, without limitation,global methods, local methods and hybrid methods, such as, e.g., segmentapproach methods. Protocols to determine percent identity are routineprocedures within the scope of one skilled in the art and from theteaching herein.

Approaches well known to one skilled in the art on how to modify aClostridial NAP in order to increase its binding activity for anendogenous Clostridial toxin receptor present on a naturally-occurringClostridial toxin target cell. As described above, one approach involvesidentifying amino acids using computational protein design algorithms;changing specifically-identified amino acids using, without limitation,site-directed mutagenesis, oligonucleotide-directed mutagenesis andsite-specific mutagenesis; and testing the binding activity of modifiedClostridial toxins comprising a modified Clostridial NAP with enhancedbinding activity using, e.g., heterogeneous assays, homogeneous assaysand non-separating homogeneous assays. It is further envisioned that thebinding activity of a modified Clostridial toxin with enhanced bindingactivity disclosed in the present specification can be determined byaffinity chromatography using immobilized receptors and interfacialoptical assays. In another approach described above, a binding activityof a modified Clostridial NAP for a naturally-occurring Clostridialtoxin receptor present on a naturally-occurring Clostridial toxin targetcell can be achieved using directed-evolution methods.

A Clostridial NAP includes, without limitation, naturally occurringClostridial NAP variants, such as, e.g., Clostridial NAP isoforms andClostridial NAP subtypes; non-naturally occurring Clostridial NAPvariants, such as, e.g., conservative Clostridial NAP variants,non-conservative Clostridial NAP variants, Clostridial NAP chimerics,active Clostridial NAP fragments thereof, or any combination thereof.

As used herein, the term “Clostridial NAP variant,” whethernaturally-occurring or non-naturally-occurring, means a Clostridial NAPthat has at least one amino acid change from the corresponding region ofthe disclosed reference sequences (see Tables 3-6) and can be describedin percent identity to the corresponding region of that referencesequence. Unless expressly indicated, all Clostridial NAP variantsdisclosed in the present specification are capable of executing the cellbinding step of the intoxication process.

It is recognized by those of skill in the art that within eachClostridial bacterium there can be naturally occurring Clostridial NAPvariants that differ somewhat in their amino acid sequence, and also inthe nucleic acids encoding these proteins. For example, there arepresently five Clostridial botulinum serotype A HA-33 variants,HA-33/A1, HA-33/A2, HA-33/A3, HA-33/A4 and HA-33/A5 (Tables 3 and 4),with specific HA-33 variants showing various degrees of amino aciddivergence when compared to another HA-33 variant. As another example,there are presently three Clostridial botulinum serotype A NTNH-33variants, NTNH/A1, NTNH/A2 and NTNH/A3 (Table 6), with specific NTNHvariant showing various degrees of amino acid divergence when comparedto another NTNH variant. As used herein, the term “naturally occurringClostridial NAP variant” means any Clostridial NAP produced by anaturally-occurring process, including, without limitation, ClostridialNAP isoforms produced from alternatively-spliced transcripts,Clostridial NAP isoforms produced by spontaneous mutation andClostridial NAP subtypes. A naturally occurring Clostridial NAP variantcan function in substantially the same manner as the referenceClostridial NAP on which the naturally occurring Clostridial NAP variantis based, and can be substituted for the reference Clostridial NAP inany aspect of the present invention. A naturally occurring ClostridialNAP variant may substitute one or more amino acids, two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, ten or more amino acids, 20 or more amino acids, 30 or moreamino acids, 40 or more amino acids, 50 or more amino acids or 100 ormore amino acids from the reference Clostridial NAP on which thenaturally occurring Clostridial NAP variant is based. A naturallyoccurring Clostridial NAP variant can also substitute at least 10contiguous amino acids, at least 15 contiguous amino acids, at least 20contiguous amino acids, or at least 25 contiguous amino acids from thereference Clostridial NAP on which the naturally occurring ClostridialNAP variant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial NAP on which thenaturally occurring Clostridial NAP variant is based.

A non-limiting examples of a naturally occurring Clostridial NAP variantis a Clostridial NAP isoform such as, e.g., a Clostridial botulinumserotype A HA-33 isoform, a Clostridial botulinum serotype B HA-33isoform, a Clostridial botulinum serotype C1 HA-33 isoform, aClostridial botulinum serotype D HA-33 isoform, a Clostridial botulinumserotype A HA-17 isoform, a Clostridial botulinum serotype B HA-17isoform, a Clostridial botulinum serotype C1 HA-17 isoform, aClostridial botulinum serotype D HA-17 isoform, a Clostridial botulinumserotype A NTNH isoform, a Clostridial botulinum serotype B NTNHisoform, a Clostridial botulinum serotype C1 NTNH isoform, a Clostridialbotulinum serotype D NTNH isoform, a Clostridial botulinum serotype ENTNH isoform, a Clostridial botulinum serotype F NTNH isoform and aClostridial botulinum serotype G NTNH isoform. A Clostridial NAP isoformcan function in substantially the same manner as the referenceClostridial NAP on which the Clostridial NAP isoform is based, and canbe substituted for the reference Clostridial NAP in any aspect of thepresent invention.

Another non-limiting examples of a naturally occurring Clostridial NAPvariant is a Clostridial NAP subtype such as, e.g., a Clostridialbotulinum serotype A HA-33 subtype, a Clostridial botulinum serotype BHA-33 subtype, a Clostridial botulinum serotype C1 HA-33 subtype, aClostridial botulinum serotype D HA-33 subtype, a Clostridial botulinumserotype A HA-17 subtype, a Clostridial botulinum serotype B HA-17subtype, a Clostridial botulinum serotype C1 HA-17 subtype, aClostridial botulinum serotype D HA-17 subtype, a Clostridial botulinumserotype A NTNH subtype, a Clostridial botulinum serotype B NTNHsubtype, a Clostridial botulinum serotype C1 NTNH subtype, a Clostridialbotulinum serotype D NTNH subtype, a Clostridial botulinum serotype ENTNH subtype, a Clostridial botulinum serotype F NTNH subtype and aClostridial botulinum serotype G NTNH subtype. A Clostridial NAP subtypecan function in substantially the same manner as the referenceClostridial NAP on which the Clostridial NAP subtype is based, and canbe substituted for the reference Clostridial NAP in any aspect of thepresent invention.

As used herein, the term “non-naturally occurring Clostridial NAPvariant” means any Clostridial NAP produced with the aid of humanmanipulation, including, without limitation, Clostridial NAPs producedby genetic engineering using random mutagenesis or rational design andClostridial NAPs produced by chemical synthesis. Non-limiting examplesof non-naturally occurring Clostridial NAP variants include, e.g.,conservative Clostridial NAP variants, non-conservative Clostridial NAPvariants, Clostridial NAP chimeric variants and active Clostridial NAPfragments.

As used herein, the term “conservative Clostridial NAP variant” means aClostridial NAP that has at least one amino acid substituted by anotheramino acid or an amino acid analog that has at least one propertysimilar to that of the original amino acid from the referenceClostridial NAP sequence (see Tables 3-6). Examples of propertiesinclude, without limitation, similar size, topography, charge,hydrophobicity, hydrophilicity, lipophilicity, covalent-bondingcapacity, hydrogen-bonding capacity, a physicochemical property, of thelike, or any combination thereof. A conservative Clostridial NAP variantcan function in substantially the same manner as the referenceClostridial NAP on which the conservative Clostridial NAP variant isbased, and can be substituted for the reference Clostridial NAP in anyaspect of the present invention. A conservative Clostridial NAP variantmay substitute one or more amino acids, two or more amino acids, threeor more amino acids, four or more amino acids, five or more amino acids,ten or more amino acids, 20 or more amino acids, 30 or more amino acids,40 or more amino acids or 50 or more amino acids from the referenceClostridial NAP on which the conservative Clostridial NAP variant isbased. A conservative Clostridial NAP variant can also substitute atleast 10 contiguous amino acids, at least 15 contiguous amino acids, atleast 20 contiguous amino acids, or at least 25 contiguous amino acidsfrom the reference Clostridial NAP on which the conservative ClostridialNAP variant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial NAP on which theconservative Clostridial NAP variant is based. Non-limiting examples ofa conservative Clostridial NAP variant include, e.g., a conservativeClostridial botulinum serotype A HA-33 variant, a conservativeClostridial botulinum serotype B HA-33 variant, a conservativeClostridial botulinum serotype C1 HA-33 variant, a conservativeClostridial botulinum serotype D HA-33 variant, a conservativeClostridial botulinum serotype A HA-17 variant, a conservativeClostridial botulinum serotype B HA-17 variant, a conservativeClostridial botulinum serotype C1 HA-17 variant, a conservativeClostridial botulinum serotype D HA-17 variant, a conservativeClostridial botulinum serotype A NTNH variant, a conservativeClostridial botulinum serotype B NTNH variant, a conservativeClostridial botulinum serotype C1 NTNH variant, a conservativeClostridial botulinum serotype D NTNH variant, a conservativeClostridial botulinum serotype E NTNH variant, a conservativeClostridial botulinum serotype F NTNH variant and a conservativeClostridial botulinum serotype G NTNH variant.

As used herein, the term “non-conservative Clostridial NAP variant”means a Clostridial NAP in which 1) at least one amino acid is deletedfrom the reference Clostridial NAP on which the non-conservativeClostridial NAP variant is based; 2) at least one amino acid added tothe reference Clostridial NAP on which the non-conservative ClostridialNAP is based; or 3) at least one amino acid is substituted by anotheramino acid or an amino acid analog that does not share any propertysimilar to that of the original amino acid from the referenceClostridial NAP sequence (see Tables 3-6). A non-conservativeClostridial NAP variant can function in substantially the same manner asthe reference Clostridial NAP on which the non-conservative ClostridialNAP variant is based, and can be substituted for the referenceClostridial NAP in any aspect of the present invention. Anon-conservative Clostridial NAP variant can delete one or more aminoacids, two or more amino acids, three or more amino acids, four or moreamino acids, five or more amino acids, and ten or more amino acids fromthe reference Clostridial NAP on which the non-conservative ClostridialNAP variant is based. A non-conservative Clostridial NAP variant can addone or more amino acids, two or more amino acids, three or more aminoacids, four or more amino acids, five or more amino acids, and ten ormore amino acids to the reference Clostridial NAP on which thenon-conservative Clostridial NAP variant is based. A non-conservativeClostridial NAP variant may substitute one or more amino acids, two ormore amino acids, three or more amino acids, four or more amino acids,five or more amino acids, ten or more amino acids, 20 or more aminoacids, 30 or more amino acids, 40 or more amino acids or 50 or moreamino acids from the reference Clostridial NAP on which thenon-conservative Clostridial NAP variant is based. A non-conservativeClostridial NAP variant can also substitute at least 10 contiguous aminoacids, at least 15 contiguous amino acids, at least 20 contiguous aminoacids, or at least 25 contiguous amino acids from the referenceClostridial NAP on which the non-conservative Clostridial NAP variant isbased, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference Clostridial NAP on which thenon-conservative Clostridial NAP variant is based. Non-limiting examplesof a non-conservative Clostridial NAP variant include, e.g., anon-conservative Clostridial botulinum serotype A HA-33 variant, anon-conservative Clostridial botulinum serotype B HA-33 variant, anon-conservative Clostridial botulinum serotype C1 HA-33 variant, anon-conservative Clostridial botulinum serotype D HA-33 variant, anon-conservative Clostridial botulinum serotype A HA-17 variant, anon-conservative Clostridial botulinum serotype B HA-17 variant, anon-conservative Clostridial botulinum serotype C1 HA-17 variant, anon-conservative Clostridial botulinum serotype D HA-17 variant, anon-conservative Clostridial botulinum serotype A NTNH variant, anon-conservative Clostridial botulinum serotype B NTNH variant, anon-conservative Clostridial botulinum serotype C1 NTNH variant, anon-conservative Clostridial botulinum serotype D NTNH variant, anon-conservative Clostridial botulinum serotype E NTNH variant, anon-conservative Clostridial botulinum serotype F NTNH variant and anon-conservative Clostridial botulinum serotype G NTNH variant.

As used herein, the term “Clostridial NAP chimeric” means a polypeptidecomprising at least a portion of a Clostridial NAP and at least aportion of at least one other polypeptide to form an enhanced targetingdomain with at least one property different from the referenceClostridial NAP (see Tables 3-6), with the proviso that this ClostridialNAP chimeric can specifically bind to a Clostridial toxin receptorpresent in a Clostridial toxin target cell, and thus participate inexecuting the overall cellular mechanism whereby a Clostridial toxinproteolytically cleaves a substrate.

As used herein, the term “active Clostridial NAP fragment” means any ofa variety of Clostridial NAP fragments comprising the enhanced targetingdomain can be useful in aspects of the present invention with theproviso that these NAP fragments can specifically bind to a Clostridialtoxin receptor present in a Clostridial toxin target cell, and thusparticipate in executing the overall cellular mechanism whereby aClostridial toxin proteolytically cleaves a substrate.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises an enhanced targeting domain comprisinga β-trefoil domain derived from a NAP. In another embodiment, a modifiedClostridial toxin disclosed in the present specification comprises anenhanced targeting domain comprising a β-trefoil domain with enhancedbinding activity derived from a NAP.

In another embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises an enhanced targeting domain comprisinga β-trefoil domain derived from a Clostridial HA-33. In an aspect ofthis embodiment, a β-trefoil domain derived from a Clostridial HA-33comprises, e.g., a β-trefoil domain derived from a Clostridial botulinumserotype A HA-33, a β-trefoil domain derived from a Clostridialbotulinum serotype B HA-33, a β-trefoil domain derived from aClostridial botulinum serotype C1 HA-33 or a β-trefoil domain derivedfrom a Clostridial botulinum serotype D HA-33. In another aspect of thisembodiment, a β-trefoil domain derived from a Clostridial HA-33comprises a 1α-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a 16-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-33, a 1γ-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33, a 2α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a 2β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype A HA-33,or a 2γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A HA-33.

In another embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises an enhanced targeting domain comprisinga β-trefoil domain with enhanced binding activity derived from aClostridial HA-33. In an aspect of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial HA-33comprises, e.g., a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype B HA-33, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype C1 HA-33 or aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-33. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial HA-33 comprises a modified 1α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a modified1β-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a modified 1γ-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a modified 2α-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33, a modified 26-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype A HA-33,or a modified 2γ-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-33 comprises a β-trefoil domain derived from aClostridial botulinum serotype A HA-33 of SEQ ID NO: 9. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype A HA-33 comprises amino acids 10-144 or amino acids 151-293 ofSEQ ID NO: 9. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype A HA-33 comprises a1α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a 16-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a 1γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-33, a 2α-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33, a 2β-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, or a2γ-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33 of SEQ ID NO: 9. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial botulinum serotype A HA-33comprises amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 9.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-33 of SEQ ID NO: 9. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises amino acids10-144 or amino acids 151-293 of SEQ ID NO: 9. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises a modified1α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a modified 16-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a modified 1γ-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33, a modified 2α-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype A HA-33,a modified 26-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, or a modified 2γ-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 9. Inanother aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 9.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:9, at least 75% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 9, at least 80% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:9, at least 85% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 9, at least 90% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:9 or at least 95% amino acid identity with amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 9. In yet other aspects ofthis embodiment, a Clostridial botulinum serotype A HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 10-54,amino acids 60-100, amino acids 105-144, amino acids 151-195, aminoacids 200-242, or amino acids 249-293 of SEQ ID NO: 9, at most 75% aminoacid identity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 9, at most 80% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9, atmost 85% amino acid identity with amino acids 10-54, amino acids 60-100,amino acids 105-144, amino acids 151-195, amino acids 200-242, or aminoacids 249-293 of SEQ ID NO: 9, at most 90% amino acid identity withamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9 orat most 95% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 9.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 9. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-100, amino acids 105-144, amino acids 151-195, aminoacids 200-242, or amino acids 249-293 of SEQ ID NO: 9. In yet otheraspects of this embodiment, a Clostridial botulinum serotype A HA-33comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 9. In other aspects of this embodiment, a Clostridial botulinumserotype A HA-33 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 9. In still other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 9. In other aspects ofthis embodiment, a Clostridial botulinum serotype A HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 9. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 9. In yet other aspects ofthis embodiment, a Clostridial botulinum serotype A HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids10-54, amino acids 60-100, amino acids 105-144, amino acids 151-195,amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9. In otheraspects of this embodiment, a Clostridial botulinum serotype A HA-33comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 9. Instill other aspects of this embodiment, a Clostridial botulinum serotypeA HA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 9. In other aspects of this embodiment, a Clostridial botulinumserotype A HA-33 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 9.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-33 comprises a 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a 1β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotype AHA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33 or a 2β8/β9 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 9. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype A HA-33 comprises amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 9.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises amodified 1β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a modified 1β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a modified2β4/β5 hairpin turn of a β-trefoil domain of a Clostridial botulinumserotype A HA-33 or a modified 2β8/β9 hairpin turn of a β-trefoil domainof a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 9. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype A HA-33 comprisesa modification of amino acids 55-59, amino acids 101-104, amino acids196-199, or amino acids 243-248 of SEQ ID NO: 9.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 9, at least 75% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 9, at least 80% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 9, at least 85% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 9, at least 90% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 9 or at least 95% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 9. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 9, at most 75% amino acid identity with amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:9, at most 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 9, atmost 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 9, atmost 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 9 orat most 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 9.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 9. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid substitutions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 9. In other aspects of this embodiment, a non-contiguousamino acid substitution of any amino acid from amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:9 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 9 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at most one, two, three orfour non-contiguous amino acid deletions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 9. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid deletions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 9. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A HA-33 comprises a polypeptide having, e.g., at mostone, two, three or four non-contiguous amino acid additions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 9. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-33 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 55-59, amino acids 101-104, aminoacids 196-199, or amino acids 243-248 of SEQ ID NO: 9.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:9. In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:9. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 9 canbe replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 9 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:9. In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid deletions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:9. In still other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at most one, two, three orfour contiguous amino acid additions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 9. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid additions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 9.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-33 comprises a β-trefoil domain derived from aClostridial botulinum serotype A HA-33 of SEQ ID NO: 10. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype A HA-33 comprises amino acids 10-144 or amino acids 151-293 ofSEQ ID NO: 10. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype A HA-33 comprises a1α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a 1β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a 1γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-33, a 2α-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33, a 2β-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, or a2γ-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33 of SEQ ID NO: 10. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial botulinum serotype A HA-33comprises amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 10.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-33 of SEQ ID NO: 10. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises amino acids10-144 or amino acids 151-293 of SEQ ID NO: 10. In another aspect ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises amodified 1α-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A HA-33, a modified 1β-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-33, a modified 1γ-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a modified2α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a modified 2β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, or a modified 2γ-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 10. Inanother aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 10.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:10, at least 75% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 10, at least 80% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 10, at least 85% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 10,at least 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 10 or at least 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 10. In yet other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-54, amino acids 60-100,amino acids 105-144, amino acids 151-195, amino acids 200-242, or aminoacids 249-293 of SEQ ID NO: 10, at most 75% amino acid identity withamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 10,at most 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 10, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:10, at most 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 10 or at most 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 10.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 10. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-100, amino acids 105-144, amino acids 151-195, aminoacids 200-242, or amino acids 249-293 of SEQ ID NO: 10. In yet otheraspects of this embodiment, a Clostridial botulinum serotype A HA-33comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 10. In other aspects of this embodiment, a Clostridial botulinumserotype A HA-33 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 10. In still other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 10. In other aspects ofthis embodiment, a Clostridial botulinum serotype A HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 10.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 10. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 10. In yet other aspectsof this embodiment, a Clostridial botulinum serotype A HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 10.In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 10. In still other aspects of this embodiment, a Clostridialbotulinum serotype A HA-33 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 10. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 10.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-33 comprises a 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a 1β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotype AHA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33 or a 2β8/β9 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 10. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype A HA-33 comprises amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 10.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises amodified 1β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a modified 1β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a modified2β4/β5 hairpin turn of a β-trefoil domain of a Clostridial botulinumserotype A HA-33 or a modified 2β8/β9 hairpin turn of a β-trefoil domainof a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 10. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype A HA-33 comprisesa modification of amino acids 55-59, amino acids 101-104, amino acids196-199, or amino acids 243-248 of SEQ ID NO: 10.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 10, at least 75% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 10, at least 80% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 10, at least 85% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 10, at least 90% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 10 or at least 95% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 10. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 10, at most 75% amino acid identity with amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:10, at most 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 10,at most 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 10,at most 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 10 orat most 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 10.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 10. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid substitutions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 10. In other aspects of this embodiment, a non-contiguousamino acid substitution of any amino acid from amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:10 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 10 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at most one, two, three orfour non-contiguous amino acid deletions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 10. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid deletions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 10. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A HA-33 comprises a polypeptide having, e.g., at mostone, two, three or four non-contiguous amino acid additions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 10. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-33 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 55-59, amino acids 101-104, aminoacids 196-199, or amino acids 243-248 of SEQ ID NO: 10.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:10. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid substitutions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 10. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 10can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 10 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:10. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid deletions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 10. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype A HA-33 comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 10. In other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A HA-33 comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid additions relativeto amino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 10.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-33 comprises a β-trefoil domain derived from aClostridial botulinum serotype A HA-33 of SEQ ID NO: 11. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype A HA-33 comprises amino acids 10-144 or amino acids 151-293 ofSEQ ID NO: 11. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype A HA-33 comprises a1α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a 1β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a 1γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-33, a 2α-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33, a 2β-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, or a2γ-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33 of SEQ ID NO: 11. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial botulinum serotype A HA-33comprises amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 11.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-33 of SEQ ID NO: 11. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises amino acids10-144 or amino acids 151-293 of SEQ ID NO: 11. In another aspect ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises amodified 1α-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A HA-33, a modified 1β-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-33, a modified 1γ-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a modified2α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a modified 2β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, or a modified 2γ-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 11. Inanother aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 11.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:11, at least 75% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 11, at least 80% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11, at least 85% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11,at least 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 11 or at least 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11. In yet other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-54, amino acids 60-100,amino acids 105-144, amino acids 151-195, amino acids 200-242, or aminoacids 249-293 of SEQ ID NO: 11, at most 75% amino acid identity withamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11,at most 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 11, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO:11, at most 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-242,or amino acids 249-293 of SEQ ID NO: 11 or at most 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-100, amino acids 105-144, amino acids 151-195, aminoacids 200-242, or amino acids 249-293 of SEQ ID NO: 11. In yet otheraspects of this embodiment, a Clostridial botulinum serotype A HA-33comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 11. In other aspects of this embodiment, a Clostridial botulinumserotype A HA-33 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11. In still other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 11. In other aspects ofthis embodiment, a Clostridial botulinum serotype A HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 11. In yet other aspectsof this embodiment, a Clostridial botulinum serotype A HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-242, or amino acids 249-293 of SEQ ID NO: 11.In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 11. In still other aspects of this embodiment, a Clostridialbotulinum serotype A HA-33 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-242, or amino acids249-293 of SEQ ID NO: 11. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-242, or amino acids 249-293 of SEQ ID NO: 11.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-33 comprises a 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a 1β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotype AHA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33 or a 2β8/β9 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 11. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype A HA-33 comprises amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 11.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises amodified 1β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a modified 1β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a modified2β4/β5 hairpin turn of a β-trefoil domain of a Clostridial botulinumserotype A HA-33 or a modified 2β8/β9 hairpin turn of a β-trefoil domainof a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 11. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype A HA-33 comprisesa modification of amino acids 55-59, amino acids 101-104, amino acids196-199, or amino acids 243-248 of SEQ ID NO: 11.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 11, at least 75% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 11, at least 80% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 11, at least 85% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 11, at least 90% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 11 or at least 95% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 11. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 11, at most 75% amino acid identity with amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:11, at most 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11,at most 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11,at most 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11 orat most 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 11. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid substitutions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 11. In other aspects of this embodiment, a non-contiguousamino acid substitution of any amino acid from amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:11 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 11 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at most one, two, three orfour non-contiguous amino acid deletions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 11. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid deletions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 11. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A HA-33 comprises a polypeptide having, e.g., at mostone, two, three or four non-contiguous amino acid additions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 11. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-33 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 55-59, amino acids 101-104, aminoacids 196-199, or amino acids 243-248 of SEQ ID NO: 11.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:11. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid substitutions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 11. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 11can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 11 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:11. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid deletions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 11. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype A HA-33 comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 11. In other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A HA-33 comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid additions relativeto amino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 11.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-33 comprises a β-trefoil domain derived from aClostridial botulinum serotype A HA-33 of SEQ ID NO: 12. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype A HA-33 comprises amino acids 10-146 or amino acids 153-294 ofSEQ ID NO: 12. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype A HA-33 comprises a1α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a 18-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a 1γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-33, a 2α-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33, a 2β-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, or a2γ-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33 of SEQ ID NO: 12. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial botulinum serotype A HA-33comprises amino acids 10-56, amino acids 62-102, amino acids 107-146,amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQID NO: 12.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-33 of SEQ ID NO: 12. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises amino acids10-146 or amino acids 153-294 of SEQ ID NO: 12. In another aspect ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises amodified 1α-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A HA-33, a modified 1β-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-33, a modified 1γ-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a modified2α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a modified 2β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, or a modified 2γ-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 12. Inanother aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises amino acids 10-56, amino acids 62-102, amino acids 107-146,amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQID NO: 12.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 10-56, amino acids 62-102, amino acids 107-146, aminoacids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO:12, at least 75% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243,or amino acids 250-294 of SEQ ID NO: 12, at least 80% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 12, at least 85% amino acid identity with aminoacids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 12,at least 90% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243,or amino acids 250-294 of SEQ ID NO: 12 or at least 95% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 12. In yet other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-56, amino acids 62-102,amino acids 107-146, amino acids 153-197, amino acids 202-243, or aminoacids 250-294 of SEQ ID NO: 12, at most 75% amino acid identity withamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 12,at most 80% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243,or amino acids 250-294 of SEQ ID NO: 12, at most 85% amino acid identitywith amino acids 10-56, amino acids 62-102, amino acids 107-146, aminoacids 153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO:12, at most 90% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 202-243,or amino acids 250-294 of SEQ ID NO: 12 or at most 95% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 12.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-56, amino acids 62-102, aminoacids 107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 12. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-56,amino acids 62-102, amino acids 107-146, amino acids 153-197, aminoacids 202-243, or amino acids 250-294 of SEQ ID NO: 12. In yet otheraspects of this embodiment, a Clostridial botulinum serotype A HA-33comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-56, amino acids 62-102, amino acids 107-146,amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQID NO: 12. In other aspects of this embodiment, a Clostridial botulinumserotype A HA-33 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 12. In still other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids202-243, or amino acids 250-294 of SEQ ID NO: 12. In other aspects ofthis embodiment, a Clostridial botulinum serotype A HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 12.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-56, amino acids 62-102, aminoacids 107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 12. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids202-243, or amino acids 250-294 of SEQ ID NO: 12. In yet other aspectsof this embodiment, a Clostridial botulinum serotype A HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 202-243, or amino acids 250-294 of SEQ ID NO: 12.In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 10-56, amino acids 62-102, amino acids 107-146,amino acids 153-197, amino acids 202-243, or amino acids 250-294 of SEQID NO: 12. In still other aspects of this embodiment, a Clostridialbotulinum serotype A HA-33 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-56, amino acids 62-102, aminoacids 107-146, amino acids 153-197, amino acids 202-243, or amino acids250-294 of SEQ ID NO: 12. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids202-243, or amino acids 250-294 of SEQ ID NO: 12.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-33 comprises a 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a 1β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotype AHA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33 or a 2β8/β9 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 12. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype A HA-33 comprises amino acids 57-61,amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQID NO: 12.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises amodified 1β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a modified 1β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a modified2β4/β5 hairpin turn of a β-trefoil domain of a Clostridial botulinumserotype A HA-33 or a modified 2β8/β9 hairpin turn of a β-trefoil domainof a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 12. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype A HA-33 comprisesa modification of amino acids 57-61, amino acids 103-106, amino acids198-201, or amino acids 244-249 of SEQ ID NO: 12.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 57-61, amino acids 103-106, amino acids 198-201, oramino acids 244-249 of SEQ ID NO: 12, at least 75% amino acid identitywith amino acids 57-61, amino acids 103-106, amino acids 198-201, oramino acids 244-249 of SEQ ID NO: 12, at least 80% amino acid identitywith amino acids 57-61, amino acids 103-106, amino acids 198-201, oramino acids 244-249 of SEQ ID NO: 12, at least 85% amino acid identitywith amino acids 57-61, amino acids 103-106, amino acids 198-201, oramino acids 244-249 of SEQ ID NO: 12, at least 90% amino acid identitywith amino acids 57-61, amino acids 103-106, amino acids 198-201, oramino acids 244-249 of SEQ ID NO: 12 or at least 95% amino acid identitywith amino acids 57-61, amino acids 103-106, amino acids 198-201, oramino acids 244-249 of SEQ ID NO: 12. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 57-61,amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQID NO: 12, at most 75% amino acid identity with amino acids 57-61, aminoacids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO:12, at most 80% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 12,at most 85% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 12,at most 90% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 12 orat most 95% amino acid identity with amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 12.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 57-61,amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQID NO: 12. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid substitutions relative to amino acids57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249of SEQ ID NO: 12. In other aspects of this embodiment, a non-contiguousamino acid substitution of any amino acid from amino acids 57-61, aminoacids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO:12 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 57-61, amino acids 103-106, amino acids 198-201, oramino acids 244-249 of SEQ ID NO: 12 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at most one, two, three orfour non-contiguous amino acid deletions relative to amino acids 57-61,amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQID NO: 12. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid deletions relative to amino acids57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249of SEQ ID NO: 12. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A HA-33 comprises a polypeptide having, e.g., at mostone, two, three or four non-contiguous amino acid additions relative toamino acids 57-61, amino acids 103-106, amino acids 198-201, or aminoacids 244-249 of SEQ ID NO: 12. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-33 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 57-61, amino acids 103-106, aminoacids 198-201, or amino acids 244-249 of SEQ ID NO: 12.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 57-61, aminoacids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO:12. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid substitutions relative to amino acids57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249of SEQ ID NO: 12. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 57-61, amino acids103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO: 12can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 57-61, amino acids 103-106, amino acids 198-201, or aminoacids 244-249 of SEQ ID NO: 12 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 57-61, aminoacids 103-106, amino acids 198-201, or amino acids 244-249 of SEQ ID NO:12. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid deletions relative to amino acids 57-61,amino acids 103-106, amino acids 198-201, or amino acids 244-249 of SEQID NO: 12. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype A HA-33 comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids57-61, amino acids 103-106, amino acids 198-201, or amino acids 244-249of SEQ ID NO: 12. In other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A HA-33 comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid additions relativeto amino acids 57-61, amino acids 103-106, amino acids 198-201, or aminoacids 244-249 of SEQ ID NO: 12.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-33 comprises a β-trefoil domain derived from aClostridial botulinum serotype A HA-33 of SEQ ID NO: 13. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype A HA-33 comprises amino acids 10-144 or amino acids 151-293 ofSEQ ID NO: 13. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype A HA-33 comprises a1α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a 1β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a 1γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-33, a 2α-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33, a 2β-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, or a2γ-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33 of SEQ ID NO: 13. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial botulinum serotype A HA-33comprises amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 13.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-33 of SEQ ID NO: 13. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises amino acids10-144 or amino acids 151-293 of SEQ ID NO: 13. In another aspect ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises amodified 1α-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A HA-33, a modified 1β-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-33, a modified 1γ-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a modified2α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeA HA-33, a modified 2β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, or a modified 2γ-fold motif of a β-trefoildomain of a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 13. Inanother aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-242, or amino acids 249-293 of SEQID NO: 13.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 10-55, amino acids 56-102, amino acids 103-144, aminoacids 151-197, amino acids 198-245, or amino acids 246-279 of SEQ ID NO:13, at least 75% amino acid identity with amino acids 10-55, amino acids56-102, amino acids 103-144, amino acids 151-197, amino acids 198-245,or amino acids 246-279 of SEQ ID NO: 13, at least 80% amino acididentity with amino acids 10-55, amino acids 56-102, amino acids103-144, amino acids 151-197, amino acids 198-245, or amino acids246-279 of SEQ ID NO: 13, at least 85% amino acid identity with aminoacids 10-55, amino acids 56-102, amino acids 103-144, amino acids151-197, amino acids 198-245, or amino acids 246-279 of SEQ ID NO: 13,at least 90% amino acid identity with amino acids 10-55, amino acids56-102, amino acids 103-144, amino acids 151-197, amino acids 198-245,or amino acids 246-279 of SEQ ID NO: 13 or at least 95% amino acididentity with amino acids 10-55, amino acids 56-102, amino acids103-144, amino acids 151-197, amino acids 198-245, or amino acids246-279 of SEQ ID NO: 13. In yet other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-55, amino acids 56-102,amino acids 103-144, amino acids 151-197, amino acids 198-245, or aminoacids 246-279 of SEQ ID NO: 13, at most 75% amino acid identity withamino acids 10-55, amino acids 56-102, amino acids 103-144, amino acids151-197, amino acids 198-245, or amino acids 246-279 of SEQ ID NO: 13,at most 80% amino acid identity with amino acids 10-55, amino acids56-102, amino acids 103-144, amino acids 151-197, amino acids 198-245,or amino acids 246-279 of SEQ ID NO: 13, at most 85% amino acid identitywith amino acids 10-55, amino acids 56-102, amino acids 103-144, aminoacids 151-197, amino acids 198-245, or amino acids 246-279 of SEQ ID NO:13, at most 90% amino acid identity with amino acids 10-55, amino acids56-102, amino acids 103-144, amino acids 151-197, amino acids 198-245,or amino acids 246-279 of SEQ ID NO: 13 or at most 95% amino acididentity with amino acids 10-55, amino acids 56-102, amino acids103-144, amino acids 151-197, amino acids 198-245, or amino acids246-279 of SEQ ID NO: 13.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a 11-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-55, amino acids 56-102, aminoacids 103-144, amino acids 151-197, amino acids 198-245, or amino acids246-279 of SEQ ID NO: 13. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-55,amino acids 56-102, amino acids 103-144, amino acids 151-197, aminoacids 198-245, or amino acids 246-279 of SEQ ID NO: 13. In yet otheraspects of this embodiment, a Clostridial botulinum serotype A HA-33comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-55, amino acids 56-102, amino acids 103-144,amino acids 151-197, amino acids 198-245, or amino acids 246-279 of SEQID NO: 13. In other aspects of this embodiment, a Clostridial botulinumserotype A HA-33 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 10-55, amino acids 56-102, amino acids103-144, amino acids 151-197, amino acids 198-245, or amino acids246-279 of SEQ ID NO: 13. In still other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-55, aminoacids 56-102, amino acids 103-144, amino acids 151-197, amino acids198-245, or amino acids 246-279 of SEQ ID NO: 13. In other aspects ofthis embodiment, a Clostridial botulinum serotype A HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 10-55, amino acids 56-102, amino acids 103-144, amino acids151-197, amino acids 198-245, or amino acids 246-279 of SEQ ID NO: 13.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-55, amino acids 56-102, aminoacids 103-144, amino acids 151-197, amino acids 198-245, or amino acids246-279 of SEQ ID NO: 13. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-55, aminoacids 56-102, amino acids 103-144, amino acids 151-197, amino acids198-245, or amino acids 246-279 of SEQ ID NO: 13. In yet other aspectsof this embodiment, a Clostridial botulinum serotype A HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-55, amino acids 56-102, amino acids 103-144, amino acids151-197, amino acids 198-245, or amino acids 246-279 of SEQ ID NO: 13.In other aspects of this embodiment, a Clostridial botulinum serotype AHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 10-55, amino acids 56-102, amino acids 103-144,amino acids 151-197, amino acids 198-245, or amino acids 246-279 of SEQID NO: 13. In still other aspects of this embodiment, a Clostridialbotulinum serotype A HA-33 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-55, amino acids 56-102, aminoacids 103-144, amino acids 151-197, amino acids 198-245, or amino acids246-279 of SEQ ID NO: 13. In other aspects of this embodiment, aClostridial botulinum serotype A HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 10-55, aminoacids 56-102, amino acids 103-144, amino acids 151-197, amino acids198-245, or amino acids 246-279 of SEQ ID NO: 13.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-33 comprises a 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a 1β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotype AHA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33 or a 2β8/β9 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 13. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype A HA-33 comprises amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 13.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-33 comprises amodified 1β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-33, a modified 1β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-33, a modified2β4/β5 hairpin turn of a β-trefoil domain of a Clostridial botulinumserotype A HA-33 or a modified 2β8/β9 hairpin turn of a β-trefoil domainof a Clostridial botulinum serotype A HA-33 of SEQ ID NO: 13. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype A HA-33 comprisesa modification of amino acids 55-59, amino acids 101-104, amino acids196-199, or amino acids 243-248 of SEQ ID NO: 13.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 13, at least 75% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 13, at least 80% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 13, at least 85% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 13, at least 90% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 13 or at least 95% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 13. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-33 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 13, at most 75% amino acid identity with amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:13, at most 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13,at most 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13,at most 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13 orat most 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 13. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid substitutions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 13. In other aspects of this embodiment, a non-contiguousamino acid substitution of any amino acid from amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:13 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 243-248 of SEQ ID NO: 13 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at most one, two, three orfour non-contiguous amino acid deletions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 13. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid deletions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 13. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A HA-33 comprises a polypeptide having, e.g., at mostone, two, three or four non-contiguous amino acid additions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 13. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-33 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 55-59, amino acids 101-104, aminoacids 196-199, or amino acids 243-248 of SEQ ID NO: 13.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:13. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid substitutions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 13. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO: 13can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 13 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 243-248 of SEQ ID NO:13. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid deletions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 243-248 of SEQID NO: 13. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype A HA-33 comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 243-248of SEQ ID NO: 13. In other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A HA-33 comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid additions relativeto amino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 243-248 of SEQ ID NO: 13.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype B HA-33 comprises a β-trefoil domain derived from aClostridial botulinum serotype B HA-33 of SEQ ID NO: 14. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype B HA-33 comprises amino acids 10-144 or amino acids 151-292 ofSEQ ID NO: 14. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype B HA-33 comprises a1α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeB HA-33, a 1β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype B HA-33, a 1γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype B HA-33, a 2α-fold motif of a β-trefoildomain of a Clostridial botulinum serotype B HA-33, a 2β-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype B HA-33, or a2γ-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeB HA-33 of SEQ ID NO: 14. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial botulinum serotype B HA-33comprises amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQID NO: 14.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B HA-33 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-33 of SEQ ID NO: 14. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype B HA-33 comprises amino acids10-144 or amino acids 151-292 of SEQ ID NO: 14. In another aspect ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B HA-33 comprises amodified 1α-fold motif of a β-trefoil domain of a Clostridial botulinumserotype B HA-33, a modified 1β-fold motif of a β-trefoil domain of aClostridial botulinum serotype B HA-33, a modified 1γ-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype B HA-33, a modified2α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeB HA-33, a modified 2β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype B HA-33, or a modified 2γ-fold motif of a β-trefoildomain of a Clostridial botulinum serotype B HA-33 of SEQ ID NO: 14. Inanother aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-33comprises amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQID NO: 14.

In other aspects of this embodiment, a Clostridial botulinum serotype BHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO:14, at least 75% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241,or amino acids 248-292 of SEQ ID NO: 14, at least 80% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 14, at least 85% amino acid identity with aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 14,at least 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241,or amino acids 248-292 of SEQ ID NO: 14 or at least 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 14. In yet other aspects of this embodiment, aClostridial botulinum serotype B HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-54, amino acids 60-100,amino acids 105-144, amino acids 151-195, amino acids 200-241, or aminoacids 248-292 of SEQ ID NO: 14, at most 75% amino acid identity withamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 14,at most 80% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241,or amino acids 248-292 of SEQ ID NO: 14, at most 85% amino acid identitywith amino acids 10-54, amino acids 60-100, amino acids 105-144, aminoacids 151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO:14, at most 90% amino acid identity with amino acids 10-54, amino acids60-100, amino acids 105-144, amino acids 151-195, amino acids 200-241,or amino acids 248-292 of SEQ ID NO: 14 or at most 95% amino acididentity with amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 14.

In other aspects of this embodiment, a Clostridial botulinum serotype BHA-33 comprising a 11-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 14. In other aspects of this embodiment, aClostridial botulinum serotype B HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-100, amino acids 105-144, amino acids 151-195, aminoacids 200-241, or amino acids 248-292 of SEQ ID NO: 14. In yet otheraspects of this embodiment, a Clostridial botulinum serotype B HA-33comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQID NO: 14. In other aspects of this embodiment, a Clostridial botulinumserotype B HA-33 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 10-54, amino acids 60-100, amino acids105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 14. In still other aspects of this embodiment, aClostridial botulinum serotype B HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-241, or amino acids 248-292 of SEQ ID NO: 14. In other aspects ofthis embodiment, a Clostridial botulinum serotype B HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 14.

In other aspects of this embodiment, a Clostridial botulinum serotype BHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 14. In other aspects of this embodiment, aClostridial botulinum serotype B HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-241, or amino acids 248-292 of SEQ ID NO: 14. In yet other aspectsof this embodiment, a Clostridial botulinum serotype B HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-100, amino acids 105-144, amino acids151-195, amino acids 200-241, or amino acids 248-292 of SEQ ID NO: 14.In other aspects of this embodiment, a Clostridial botulinum serotype BHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-100, amino acids 105-144,amino acids 151-195, amino acids 200-241, or amino acids 248-292 of SEQID NO: 14. In still other aspects of this embodiment, a Clostridialbotulinum serotype B HA-33 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-54, amino acids 60-100, aminoacids 105-144, amino acids 151-195, amino acids 200-241, or amino acids248-292 of SEQ ID NO: 14. In other aspects of this embodiment, aClostridial botulinum serotype B HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-100, amino acids 105-144, amino acids 151-195, amino acids200-241, or amino acids 248-292 of SEQ ID NO: 14.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype B HA-33 comprises a 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype B HA-33, a 1β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotype BHA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype B HA-33 or a 2β8/β9 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype B HA-33 of SEQ ID NO: 14. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype B HA-33 comprises amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQID NO: 14.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B HA-33 comprises amodified 1β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype B HA-33, a modified 1β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype B HA-33, a modified2β4/β5 hairpin turn of a β-trefoil domain of a Clostridial botulinumserotype B HA-33 or a modified 2β8/β9 hairpin turn of a β-trefoil domainof a Clostridial botulinum serotype B HA-33 of SEQ ID NO: 14. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype B HA-33 comprisesa modification of amino acids 55-59, amino acids 101-104, amino acids196-199, or amino acids 242-247 of SEQ ID NO: 14.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-33comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 242-247 of SEQ ID NO: 14, at least 75% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 242-247 of SEQ ID NO: 14, at least 80% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 242-247 of SEQ ID NO: 14, at least 85% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 242-247 of SEQ ID NO: 14, at least 90% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 242-247 of SEQ ID NO: 14 or at least 95% amino acid identitywith amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 242-247 of SEQ ID NO: 14. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype B HA-33 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQID NO: 14, at most 75% amino acid identity with amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO:14, at most 80% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 14,at most 85% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 14,at most 90% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 14 orat most 95% amino acid identity with amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 14.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-33comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQID NO: 14. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid substitutions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247of SEQ ID NO: 14. In other aspects of this embodiment, a non-contiguousamino acid substitution of any amino acid from amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO:14 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 101-104, amino acids 196-199, oramino acids 242-247 of SEQ ID NO: 14 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-33 comprises a polypeptide having, e.g., at most one, two, three orfour non-contiguous amino acid deletions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQID NO: 14. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid deletions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247of SEQ ID NO: 14. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype B HA-33 comprises a polypeptide having, e.g., at mostone, two, three or four non-contiguous amino acid additions relative toamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 242-247 of SEQ ID NO: 14. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-33 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 55-59, amino acids 101-104, aminoacids 196-199, or amino acids 242-247 of SEQ ID NO: 14.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO:14. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid substitutions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247of SEQ ID NO: 14. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 55-59, amino acids101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO: 14can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 242-247 of SEQ ID NO: 14 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 55-59, aminoacids 101-104, amino acids 196-199, or amino acids 242-247 of SEQ ID NO:14. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid deletions relative to amino acids 55-59,amino acids 101-104, amino acids 196-199, or amino acids 242-247 of SEQID NO: 14. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype B HA-33 comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids55-59, amino acids 101-104, amino acids 196-199, or amino acids 242-247of SEQ ID NO: 14. In other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype B HA-33 comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid additions relativeto amino acids 55-59, amino acids 101-104, amino acids 196-199, or aminoacids 242-247 of SEQ ID NO: 14.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B HA-33 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-33 of SEQ ID NO: 15. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype B HA-33 comprisesamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 15.

In other aspects of this embodiment, a Clostridial botulinum serotype BHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 10-56, amino acids 62-102, amino acids 107-146, aminoacids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO:15, at least 75% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242,or amino acids 249-291 of SEQ ID NO: 15, at least 80% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 15, at least 85% amino acid identity with aminoacids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 15,at least 90% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242,or amino acids 249-291 of SEQ ID NO: 15 or at least 95% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 15. In yet other aspects of this embodiment, aClostridial botulinum serotype B HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost 70% amino acid identity with amino acids 10-56, amino acids 62-102,amino acids 107-146, amino acids 153-197, amino acids 200-242, or aminoacids 249-291 of SEQ ID NO: 15, at most 75% amino acid identity withamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 15,at most 80% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242,or amino acids 249-291 of SEQ ID NO: 15, at most 85% amino acid identitywith amino acids 10-56, amino acids 62-102, amino acids 107-146, aminoacids 153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO:15, at most 90% amino acid identity with amino acids 10-56, amino acids62-102, amino acids 107-146, amino acids 153-197, amino acids 200-242,or amino acids 249-291 of SEQ ID NO: 15 or at most 95% amino acididentity with amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 15.

In other aspects of this embodiment, a Clostridial botulinum serotype BHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-56, amino acids 62-102, aminoacids 107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 15. In other aspects of this embodiment, aClostridial botulinum serotype B HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-56,amino acids 62-102, amino acids 107-146, amino acids 153-197, aminoacids 200-242, or amino acids 249-291 of SEQ ID NO: 15. In yet otheraspects of this embodiment, a Clostridial botulinum serotype B HA-33comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-56, amino acids 62-102, amino acids 107-146,amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQID NO: 15. In other aspects of this embodiment, a Clostridial botulinumserotype B HA-33 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 10-56, amino acids 62-102, amino acids107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 15. In still other aspects of this embodiment, aClostridial botulinum serotype B HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids200-242, or amino acids 249-291 of SEQ ID NO: 15. In other aspects ofthis embodiment, a Clostridial botulinum serotype B HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 15.

In other aspects of this embodiment, a Clostridial botulinum serotype BHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-56, amino acids 62-102, aminoacids 107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 15. In other aspects of this embodiment, aClostridial botulinum serotype B HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids200-242, or amino acids 249-291 of SEQ ID NO: 15. In yet other aspectsof this embodiment, a Clostridial botulinum serotype B HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-56, amino acids 62-102, amino acids 107-146, amino acids153-197, amino acids 200-242, or amino acids 249-291 of SEQ ID NO: 15.In other aspects of this embodiment, a Clostridial botulinum serotype BHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 10-56, amino acids 62-102, amino acids 107-146,amino acids 153-197, amino acids 200-242, or amino acids 249-291 of SEQID NO: 15. In still other aspects of this embodiment, a Clostridialbotulinum serotype B HA-33 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-56, amino acids 62-102, aminoacids 107-146, amino acids 153-197, amino acids 200-242, or amino acids249-291 of SEQ ID NO: 15. In other aspects of this embodiment, aClostridial botulinum serotype B HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 10-56, aminoacids 62-102, amino acids 107-146, amino acids 153-197, amino acids200-242, or amino acids 249-291 of SEQ ID NO: 15.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype B HA-33 comprises a 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype B HA-33, a 1β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotype BHA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype B HA-33 or a 2β8/β9 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype B HA-33 of SEQ ID NO: 15. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype B HA-33 comprises amino acids 56-61,amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQID NO: 15.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B HA-33 comprises amodified 1β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype B HA-33, a modified 1β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype B HA-33, a modified2β4/β5 hairpin turn of a β-trefoil domain of a Clostridial botulinumserotype B HA-33 or a modified 2β8/β9 hairpin turn of a β-trefoil domainof a Clostridial botulinum serotype B HA-33 of SEQ ID NO: 15. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype B HA-33 comprisesa modification of amino acids 56-61, amino acids 103-106, amino acids198-201, or amino acids 243-248 of SEQ ID NO: 15.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-33comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 56-61, amino acids 103-106, amino acids 198-201, oramino acids 243-248 of SEQ ID NO: 15, at least 75% amino acid identitywith amino acids 56-61, amino acids 103-106, amino acids 198-201, oramino acids 243-248 of SEQ ID NO: 15, at least 80% amino acid identitywith amino acids 56-61, amino acids 103-106, amino acids 198-201, oramino acids 243-248 of SEQ ID NO: 15, at least 85% amino acid identitywith amino acids 56-61, amino acids 103-106, amino acids 198-201, oramino acids 243-248 of SEQ ID NO: 15, at least 90% amino acid identitywith amino acids 56-61, amino acids 103-106, amino acids 198-201, oramino acids 243-248 of SEQ ID NO: 15 or at least 95% amino acid identitywith amino acids 56-61, amino acids 103-106, amino acids 198-201, oramino acids 243-248 of SEQ ID NO: 15. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype B HA-33 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 56-61,amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQID NO: 15, at most 75% amino acid identity with amino acids 56-61, aminoacids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO:15, at most 80% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 15,at most 85% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 15,at most 90% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 15 orat most 95% amino acid identity with amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 15.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-33comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 56-61,amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQID NO: 15. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid substitutions relative to amino acids56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248of SEQ ID NO: 15. In other aspects of this embodiment, a non-contiguousamino acid substitution of any amino acid from amino acids 56-61, aminoacids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO:15 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 56-61, amino acids 103-106, amino acids 198-201, oramino acids 243-248 of SEQ ID NO: 15 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-33 comprises a polypeptide having, e.g., at most one, two, three orfour non-contiguous amino acid deletions relative to amino acids 56-61,amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQID NO: 15. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid deletions relative to amino acids56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248of SEQ ID NO: 15. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype B HA-33 comprises a polypeptide having, e.g., at mostone, two, three or four non-contiguous amino acid additions relative toamino acids 56-61, amino acids 103-106, amino acids 198-201, or aminoacids 243-248 of SEQ ID NO: 15. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-33 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 56-61, amino acids 103-106, aminoacids 198-201, or amino acids 243-248 of SEQ ID NO: 15.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 56-61, aminoacids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO:15. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid substitutions relative to amino acids56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248of SEQ ID NO: 15. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 56-61, amino acids103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO: 15can be replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 56-61, amino acids 103-106, amino acids 198-201, or aminoacids 243-248 of SEQ ID NO: 15 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 56-61, aminoacids 103-106, amino acids 198-201, or amino acids 243-248 of SEQ ID NO:15. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid deletions relative to amino acids 56-61,amino acids 103-106, amino acids 198-201, or amino acids 243-248 of SEQID NO: 15. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype B HA-33 comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids56-61, amino acids 103-106, amino acids 198-201, or amino acids 243-248of SEQ ID NO: 15. In other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype B HA-33 comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid additions relativeto amino acids 56-61, amino acids 103-106, amino acids 198-201, or aminoacids 243-248 of SEQ ID NO: 15.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype C1 HA-33 comprises a β-trefoil domain derived from aClostridial botulinum serotype C1 HA-33 of SEQ ID NO: 16. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype C1 HA-33 comprises amino acids 10-141 or amino acids 148-285 ofSEQ ID NO: 16. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype C1 HA-33 comprises a1αfold motif of a β-trefoil domain of a Clostridial botulinum serotypeC1 HA-33, a 1β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-33, a 1γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype C1 HA-33, a 2α-fold motif of a β-trefoildomain of a Clostridial botulinum serotype C1 HA-33, a 2β-fold motif ofa β-trefoil domain of a Clostridial botulinum serotype C1 HA-33, or a2γ-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeC1 HA-33 of SEQ ID NO: 16. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial botulinum serotype C1 HA-33comprises amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQID NO: 16.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-33 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-33 of SEQ ID NO: 16. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 HA-33 comprises amino acids10-141 or amino acids 148-285 of SEQ ID NO: 16. In another aspect ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-33 comprises amodified 1α-fold motif of a β-trefoil domain of a Clostridial botulinumserotype C1 HA-33, a modified 1β-fold motif of a β-trefoil domain of aClostridial botulinum serotype C1 HA-33, a modified 1γ-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-33, amodified 2α-fold motif of a β-trefoil domain of a Clostridial botulinumserotype C1 HA-33, a modified 2β-fold motif of a β-trefoil domain of aClostridial botulinum serotype C1 HA-33, or a modified 2γ-fold motif ofa β-trefoil domain of a Clostridial botulinum serotype C1 HA-33 of SEQID NO: 16. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises amino acids 10-54, amino acids 60-98, amino acids103-141, amino acids 148-190, amino acids 195-234, or amino acids241-285 of SEQ ID NO: 16.

In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO:16, at least 75% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, oramino acids 241-285 of SEQ ID NO: 16, at least 80% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO:16, at least 85% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, oramino acids 241-285 of SEQ ID NO: 16, at least 90% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO:16 or at least 95% amino acid identity with amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-234, or amino acids 241-285 of SEQ ID NO: 16. In yet other aspectsof this embodiment, a Clostridial botulinum serotype C1 HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 16,at most 75% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, oramino acids 241-285 of SEQ ID NO: 16, at most 80% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO:16, at most 85% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-234, oramino acids 241-285 of SEQ ID NO: 16, at most 90% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO:16 or at most 95% amino acid identity with amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-234, or amino acids 241-285 of SEQ ID NO: 16.

In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-234, or amino acids241-285 of SEQ ID NO: 16. In other aspects of this embodiment, aClostridial botulinum serotype C1 HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-234, or amino acids 241-285 of SEQ ID NO: 16. In yet other aspectsof this embodiment, a Clostridial botulinum serotype C1 HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQID NO: 16. In other aspects of this embodiment, a Clostridial botulinumserotype C1 HA-33 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 10-54, amino acids 60-98, amino acids103-141, amino acids 148-190, amino acids 195-234, or amino acids241-285 of SEQ ID NO: 16. In still other aspects of this embodiment, aClostridial botulinum serotype C1 HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-234, or amino acids 241-285 of SEQ ID NO: 16. In other aspects ofthis embodiment, a Clostridial botulinum serotype C1 HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 16.

In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-234, or amino acids241-285 of SEQ ID NO: 16. In other aspects of this embodiment, aClostridial botulinum serotype C1 HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-234, or amino acids 241-285 of SEQ ID NO: 16. In yet other aspectsof this embodiment, a Clostridial botulinum serotype C1 HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-234, or amino acids 241-285 of SEQ ID NO: 16.In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-234, or amino acids 241-285 of SEQID NO: 16. In still other aspects of this embodiment, a Clostridialbotulinum serotype C1 HA-33 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-234, or amino acids241-285 of SEQ ID NO: 16. In other aspects of this embodiment, aClostridial botulinum serotype C1 HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-234, or amino acids 241-285 of SEQ ID NO: 16.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype C1 HA-33 comprises a 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-33, a 1β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotypeC1 HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-33 or a 2β8/β9 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype C1 HA-33 of SEQ ID NO: 16. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype C1 HA-33 comprises amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQID NO: 16.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-33 comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-33 or a modified 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-33, amodified 1β8/β9 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-33, a modified 2β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-33 or amodified 2β8/β9 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-33 of SEQ ID NO: 16. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 HA-33 comprises a modificationof amino acids 55-59, amino acids 99-102, amino acids 191-194, or aminoacids 235-240 of SEQ ID NO: 16.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-33comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 235-240 of SEQ ID NO: 16, at least 75% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 235-240 of SEQ ID NO: 16, at least 80% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 235-240 of SEQ ID NO: 16, at least 85% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 235-240 of SEQ ID NO: 16, at least 90% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 235-240 of SEQ ID NO: 16 or at least 95% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 235-240 of SEQ ID NO: 16. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 HA-33 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQID NO: 16, at most 75% amino acid identity with amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO:16, at most 80% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 16, atmost 85% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 16, at most90% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 16 or at most95% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 16.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-33comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQID NO: 16. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid substitutions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240of SEQ ID NO: 16. In other aspects of this embodiment, a non-contiguousamino acid substitution of any amino acid from amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO:16 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 235-240 of SEQ ID NO: 16 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises a polypeptide having, e.g., at most one, two, threeor four non-contiguous amino acid deletions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240of SEQ ID NO: 16. In other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype C1 HA-33 comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid deletionsrelative to amino acids 55-59, amino acids 99-102, amino acids 191-194,or amino acids 235-240 of SEQ ID NO: 16. In still other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 HA-33 comprises a polypeptidehaving, e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 55-59, amino acids 99-102, amino acids191-194, or amino acids 235-240 of SEQ ID NO: 16. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-33 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid additions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO:16.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO:16. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid substitutions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 235-240of SEQ ID NO: 16. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO: 16 canbe replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 99-102, amino acids 191-194, or aminoacids 235-240 of SEQ ID NO: 16 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 235-240 of SEQ ID NO:16. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid deletions relative to amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 235-240 of SEQID NO: 16. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype C1 HA-33 comprises a polypeptide having, e.g., at most one,two, three or four contiguous amino acid additions relative to aminoacids 55-59, amino acids 99-102, amino acids 191-194, or amino acids235-240 of SEQ ID NO: 16. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-33 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acid additionsrelative to amino acids 55-59, amino acids 99-102, amino acids 191-194,or amino acids 235-240 of SEQ ID NO: 16.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype C1 HA-33 comprises a β-trefoil domain derived from aClostridial botulinum serotype C1 HA-33 of SEQ ID NO: 17. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype C1 HA-33 comprises amino acids 10-141 or amino acids 148-286 ofSEQ ID NO: 17. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype C1 HA-33 comprises a1α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeC1 HA-33, a 1β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-33, a 1γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype C1 HA-33, a 2α-fold motif of a β-trefoildomain of a Clostridial botulinum serotype C1 HA-33, a 2β-fold motif ofa β-trefoil domain of a Clostridial botulinum serotype C1 HA-33, or a2γ-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeC1 HA-33 of SEQ ID NO: 17. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial botulinum serotype C1 HA-33comprises amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQID NO: 17.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-33 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-33 of SEQ ID NO: 17. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 HA-33 comprises amino acids10-141 or amino acids 148-286 of SEQ ID NO: 17. In another aspect ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-33 comprises amodified 1α-fold motif of a β-trefoil domain of a Clostridial botulinumserotype C1 HA-33, a modified 1β-fold motif of a β-trefoil domain of aClostridial botulinum serotype C1 HA-33, a modified 1γ-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-33, amodified 2α-fold motif of a β-trefoil domain of a Clostridial botulinumserotype C1 HA-33, a modified 2β-fold motif of a β-trefoil domain of aClostridial botulinum serotype C1 HA-33, or a modified 2γ-fold motif ofa β-trefoil domain of a Clostridial botulinum serotype C1 HA-33 of SEQID NO: 17. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises amino acids 10-54, amino acids 60-98, amino acids103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 17.

In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-33 comprising a 11-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:17, at least 75% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 17, at least 80% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:17, at least 85% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 17, at least 90% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:17 or at least 95% amino acid identity with amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 17. In yet other aspectsof this embodiment, a Clostridial botulinum serotype C1 HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 17,at most 75% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 17, at most 80% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:17, at most 85% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 17, at most 90% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:17 or at most 95% amino acid identity with amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 17.

In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 17. In other aspects of this embodiment, aClostridial botulinum serotype C1 HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 17. In yet other aspectsof this embodiment, a Clostridial botulinum serotype C1 HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQID NO: 17. In other aspects of this embodiment, a Clostridial botulinumserotype C1 HA-33 comprising a 11-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 10-54, amino acids 60-98, amino acids103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 17. In still other aspects of this embodiment, aClostridial botulinum serotype C1 HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 17. In other aspects ofthis embodiment, a Clostridial botulinum serotype C1 HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 17.

In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 17. In other aspects of this embodiment, aClostridial botulinum serotype C1 HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 17. In yet other aspectsof this embodiment, a Clostridial botulinum serotype C1 HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 17.In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQID NO: 17. In still other aspects of this embodiment, a Clostridialbotulinum serotype C1 HA-33 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 17. In other aspects of this embodiment, aClostridial botulinum serotype C1 HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 17.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype C1 HA-33 comprises a 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-33, a 1β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotypeC1 HA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-33 or a 2β8/β9 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype C1 HA-33 of SEQ ID NO: 17. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype C1 HA-33 comprises amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 17.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-33 comprises amodified 1β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-33, a modified 1β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-33, amodified 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-33 or a modified 2β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-33 of SEQ IDNO: 17. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises a modification of amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 17.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-33comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 17, at least 75% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 17, at least 80% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 17, at least 85% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 17, at least 90% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 17 or at least 95% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 17. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 HA-33 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 17, at most 75% amino acid identity with amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:17, at most 80% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 17, atmost 85% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 17, at most90% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 17 or at most95% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 17.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-33comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 17. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid substitutions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 17. In other aspects of this embodiment, a non-contiguousamino acid substitution of any amino acid from amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:17 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 17 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises a polypeptide having, e.g., at most one, two, threeor four non-contiguous amino acid deletions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 17. In other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype C1 HA-33 comprises a polypeptide having, e.g., atleast one, two, three or four non-contiguous amino acid deletionsrelative to amino acids 55-59, amino acids 99-102, amino acids 191-194,or amino acids 236-241 of SEQ ID NO: 17. In still other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 HA-33 comprises a polypeptidehaving, e.g., at most one, two, three or four non-contiguous amino acidadditions relative to amino acids 55-59, amino acids 99-102, amino acids191-194, or amino acids 236-241 of SEQ ID NO: 17. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-33 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid additions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:17.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:17. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid substitutions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 17. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 17 canbe replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 99-102, amino acids 191-194, or aminoacids 236-241 of SEQ ID NO: 17 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:17. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid deletions relative to amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 17. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype C1 HA-33 comprises a polypeptide having, e.g., at most one,two, three or four contiguous amino acid additions relative to aminoacids 55-59, amino acids 99-102, amino acids 191-194, or amino acids236-241 of SEQ ID NO: 17. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-33 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acid additionsrelative to amino acids 55-59, amino acids 99-102, amino acids 191-194,or amino acids 236-241 of SEQ ID NO: 17.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype D HA-33 comprises a β-trefoil domain derived from aClostridial botulinum serotype D HA-33 of SEQ ID NO: 18. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype D HA-33 comprises amino acids 10-141 or amino acids 148-286 ofSEQ ID NO: 18. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype D HA-33 comprises a1α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeD HA-33, a 1β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype D HA-33, a 1γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype D HA-33, a 2α-fold motif of a β-trefoildomain of a Clostridial botulinum serotype D HA-33, a 2β-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype D HA-33, or a2γ-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeD HA-33 of SEQ ID NO: 18. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial botulinum serotype D HA-33comprises amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQID NO: 18.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype D HA-33 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-33 of SEQ ID NO: 18. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype D HA-33 comprises amino acids10-141 or amino acids 148-286 of SEQ ID NO: 18. In another aspect ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype D HA-33 comprises amodified 1α-fold motif of a β-trefoil domain of a Clostridial botulinumserotype D HA-33, a modified 1β-fold motif of a β-trefoil domain of aClostridial botulinum serotype D HA-33, a modified 1γ-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype D HA-33, a modified2α-fold motif of a β-trefoil domain of a Clostridial botulinum serotypeD HA-33, a modified 2β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype D HA-33, or a modified 2γ-fold motif of a β-trefoildomain of a Clostridial botulinum serotype D HA-33 of SEQ ID NO: 18. Inanother aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D HA-33comprises amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQID NO: 18.

In other aspects of this embodiment, a Clostridial botulinum serotype DHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:18, at least 75% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 18, at least 80% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:18, at least 85% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 18, at least 90% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:18 or at least 95% amino acid identity with amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 18. In yet other aspectsof this embodiment, a Clostridial botulinum serotype D HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 18,at most 75% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 18, at most 80% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:18, at most 85% amino acid identity with amino acids 10-54, amino acids60-98, amino acids 103-141, amino acids 148-190, amino acids 195-235, oramino acids 242-286 of SEQ ID NO: 18, at most 90% amino acid identitywith amino acids 10-54, amino acids 60-98, amino acids 103-141, aminoacids 148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO:18 or at most 95% amino acid identity with amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 18.

In other aspects of this embodiment, a Clostridial botulinum serotype DHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 18. In other aspects of this embodiment, aClostridial botulinum serotype D HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 10-54,amino acids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 18. In yet other aspectsof this embodiment, a Clostridial botulinum serotype D HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQID NO: 18. In other aspects of this embodiment, a Clostridial botulinumserotype D HA-33 comprising a 11-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino aciddeletions relative to amino acids 10-54, amino acids 60-98, amino acids103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 18. In still other aspects of this embodiment, aClostridial botulinum serotype D HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 18. In other aspects ofthis embodiment, a Clostridial botulinum serotype D HA-33 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 18.

In other aspects of this embodiment, a Clostridial botulinum serotype DHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 18. In other aspects of this embodiment, aClostridial botulinum serotype D HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 18. In yet other aspectsof this embodiment, a Clostridial botulinum serotype D HA-33 comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 10-54, amino acids 60-98, amino acids 103-141, amino acids148-190, amino acids 195-235, or amino acids 242-286 of SEQ ID NO: 18.In other aspects of this embodiment, a Clostridial botulinum serotype DHA-33 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid deletionsrelative to amino acids 10-54, amino acids 60-98, amino acids 103-141,amino acids 148-190, amino acids 195-235, or amino acids 242-286 of SEQID NO: 18. In still other aspects of this embodiment, a Clostridialbotulinum serotype D HA-33 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 10-54, amino acids 60-98, aminoacids 103-141, amino acids 148-190, amino acids 195-235, or amino acids242-286 of SEQ ID NO: 18. In other aspects of this embodiment, aClostridial botulinum serotype D HA-33 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid additions relative to amino acids 10-54, aminoacids 60-98, amino acids 103-141, amino acids 148-190, amino acids195-235, or amino acids 242-286 of SEQ ID NO: 18.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype D HA-33 comprises a 1β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype D HA-33, a 1β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotype DHA-33, a 2β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype D HA-33 or a 2β8/β9 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype D HA-33 of SEQ ID NO: 18. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype D HA-33 comprises amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 18.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype D HA-33 comprises amodified 1β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype D HA-33, a modified 1β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype D HA-33, a modified2β4/β5 hairpin turn of a β-trefoil domain of a Clostridial botulinumserotype D HA-33 or a modified 2β8/β9 hairpin turn of a β-trefoil domainof a Clostridial botulinum serotype D HA-33 of SEQ ID NO: 18. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype D HA-33 comprisesa modification of amino acids 55-59, amino acids 99-102, amino acids191-194, or amino acids 236-241 of SEQ ID NO: 18.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D HA-33comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 18, at least 75% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 18, at least 80% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 18, at least 85% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 18, at least 90% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 18 or at least 95% amino acid identitywith amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 18. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype D HA-33 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 18, at most 75% amino acid identity with amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:18, at most 80% amino acid identity with amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 18, atmost 85% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 18, at most90% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 18 or at most95% amino acid identity with amino acids 55-59, amino acids 99-102,amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 18.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D HA-33comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 18. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeD HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid substitutions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 18. In other aspects of this embodiment, a non-contiguousamino acid substitution of any amino acid from amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:18 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 55-59, amino acids 99-102, amino acids 191-194, oramino acids 236-241 of SEQ ID NO: 18 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeD HA-33 comprises a polypeptide having, e.g., at most one, two, three orfour non-contiguous amino acid deletions relative to amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 18. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeD HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four non-contiguous amino acid deletions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 18. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype D HA-33 comprises a polypeptide having, e.g., at mostone, two, three or four non-contiguous amino acid additions relative toamino acids 55-59, amino acids 99-102, amino acids 191-194, or aminoacids 236-241 of SEQ ID NO: 18. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-33 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 55-59, amino acids 99-102, amino acids191-194, or amino acids 236-241 of SEQ ID NO: 18.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:18. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeD HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid substitutions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 18. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 55-59, amino acids99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO: 18 canbe replaced with glycine. In other aspects of this embodiment,contiguous amino acid substitutions of hydrophobic amino acids fromamino acids 55-59, amino acids 99-102, amino acids 191-194, or aminoacids 236-241 of SEQ ID NO: 18 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D HA-33comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 55-59, aminoacids 99-102, amino acids 191-194, or amino acids 236-241 of SEQ ID NO:18. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeD HA-33 comprises a polypeptide having, e.g., at least one, two, threeor four contiguous amino acid deletions relative to amino acids 55-59,amino acids 99-102, amino acids 191-194, or amino acids 236-241 of SEQID NO: 18. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype D HA-33 comprises a polypeptide having, e.g., at most one, two,three or four contiguous amino acid additions relative to amino acids55-59, amino acids 99-102, amino acids 191-194, or amino acids 236-241of SEQ ID NO: 18. In other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype D HA-33 comprises a polypeptide having, e.g., atleast one, two, three or four contiguous amino acid additions relativeto amino acids 55-59, amino acids 99-102, amino acids 191-194, or aminoacids 236-241 of SEQ ID NO: 18.

In an embodiment, a modified Clostridial toxin disclosed in the presentspecification comprises an enhanced targeting domain comprising aβ-trefoil domain derived from a Clostridial HA-17. In an aspect of thisembodiment, a β-trefoil domain derived from a Clostridial HA-17comprises, e.g., a β-trefoil domain derived from a Clostridial botulinumserotype A HA-17, a β-trefoil domain derived from a Clostridialbotulinum serotype B HA-17, a β-trefoil domain derived from aClostridial botulinum serotype C1 HA-17 or a β-trefoil domain derivedfrom a Clostridial botulinum serotype D HA-17. In another aspect of thisembodiment, a β-trefoil domain derived from a Clostridial HA-17comprises a α-fold motif of a β-trefoil domain of a Clostridial HA-17, aβ-fold motif of a β-trefoil domain of a Clostridial HA-17 or a γ-foldmotif of a β-trefoil domain of a Clostridial HA-17.

In an embodiment, a modified Clostridial toxin disclosed in the presentspecification comprises an enhanced targeting domain comprising aβ-trefoil domain with enhanced binding activity derived from aClostridial HA-17. In an aspect of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial HA-17comprises, e.g., a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-17, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype B HA-17, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype C1 HA-17 or aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-17. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial HA-17 comprises a modified α-fold motif of aβ-trefoil domain of a Clostridial HA-17, a modified β-fold motif of aβ-trefoil domain of a Clostridial HA-17 or a modified γ-fold motif of aβ-trefoil domain of a Clostridial HA-17.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-17 comprises a β-trefoil domain derived from aClostridial botulinum serotype A HA-17 of SEQ ID NO: 19. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype A HA-17 comprises amino acids 9-146 of SEQ ID NO: 19. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype A HA-17 comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A HA-17, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype A HA-17or a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A HA-17 of SEQ ID NO: 19. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype A HA-17comprises amino acids 9-50, amino acids 55-91, or amino acids 95-146 ofSEQ ID NO: 19.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-17 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-17 of SEQ ID NO: 19. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-17 comprises amino acids9-146 of SEQ ID NO: 19. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-17 comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype A HA-17, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A HA-17 or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype A HA-17 of SEQ ID NO: 19. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype A HA-17 comprisesamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:19.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQID NO: 19, at least 75% amino acid identity with amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 19, at least 80% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 19, at least 85% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 19,at least 90% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 19 or at least 95% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 19. In yet other aspects of this embodiment, a Clostridialbotulinum serotype A HA-17 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 19, at most 75% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 19,at most 80% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 19, at most 85% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 19, at most 90% amino acid identity with amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 19 or at most 95%amino acid identity with amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 19.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 19. In other aspects of this embodiment, aClostridial botulinum serotype A HA-17 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 19. In yet otheraspects of this embodiment, a Clostridial botulinum serotype A HA-17comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 19. In other aspects of this embodiment, a Clostridialbotulinum serotype A HA-17 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 9-50, amino acids 55-91, oramino acids 95-146 of SEQ ID NO: 19. In still other aspects of thisembodiment, a Clostridial botulinum serotype A HA-17 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 19.In other aspects of this embodiment, a Clostridial botulinum serotype AHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 19.

In other aspects of this embodiment, a Clostridial botulinum serotype AHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 19. In other aspects of this embodiment, aClostridial botulinum serotype A HA-17 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 19. In yet otheraspects of this embodiment, a Clostridial botulinum serotype A HA-17comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:19. In other aspects of this embodiment, a Clostridial botulinumserotype A HA-17 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 19. In still other aspects of thisembodiment, a Clostridial botulinum serotype A HA-17 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 19. Inother aspects of this embodiment, a Clostridial botulinum serotype AHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 19.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A HA-17 comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype A HA-17 or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype A HA-17of SEQ ID NO: 19. In another aspect of this embodiment, a β-trefoildomain derived from a Clostridial botulinum serotype A HA-17 comprisesamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 19.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-17 comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A HA-17 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A HA-17 of SEQ IDNO: 19. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA HA-17 comprises a modification of amino acids 51-54 or amino acids92-94 of SEQ ID NO: 19.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-17comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 19, at least75% amino acid identity with amino acids 51-54 or amino acids 92-94 ofSEQ ID NO: 19, at least 80% amino acid identity with amino acids 51-54or amino acids 92-94 of SEQ ID NO: 19, at least 85% amino acid identitywith amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 19, at least90% amino acid identity with amino acids 51-54 or amino acids 92-94 ofSEQ ID NO: 19 or at least 95% amino acid identity with amino acids 51-54or amino acids 92-94 of SEQ ID NO: 19. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A HA-17 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 19, at most 75% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 19, at most 80%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 19, at most 85% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 19, at most 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 19 or at most 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 19.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-17comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 19. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-17 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 51-54 or amino acids 92-94 of SEQID NO: 19. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 19 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 51-54 or amino acids 92-94of SEQ ID NO: 19 can be replaced with phenylalanine. In yet otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype A HA-17 comprisesa polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 19. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-17 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 19. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype A HA-17 comprises a polypeptide having, e.g., at most one, two,three or four non-contiguous amino acid additions relative to aminoacids 51-54 or amino acids 92-94 of SEQ ID NO: 19. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A HA-17 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid additions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 19.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-17comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 19. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-17 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 51-54 or amino acids 92-94 of SEQID NO: 19. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 51-54 or amino acids 92-94of SEQ ID NO: 19 can be replaced with glycine. In other aspects of thisembodiment, contiguous amino acid substitutions of hydrophobic aminoacids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 19 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-17 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 19. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-17comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid deletions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 19. In still other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A HA-17 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid additionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 19. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A HA-17comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 19.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype B HA-17 comprises a β-trefoil domain derived from aClostridial botulinum serotype B HA-17 of SEQ ID NO: 20. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype B HA-17 comprises amino acids 9-146 of SEQ ID NO: 20. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype B HA-17 comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype B HA-17, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype B HA-17or a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype B HA-17 of SEQ ID NO: 20. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype B HA-17comprises amino acids 9-50, amino acids 55-91, or amino acids 95-146 ofSEQ ID NO: 20.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B HA-17 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-17 of SEQ ID NO: 20. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype B HA-17 comprises amino acids9-146 of SEQ ID NO: 20. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-17 comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype B HA-17, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype B HA-17 or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype B HA-17 of SEQ ID NO: 20. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype B HA-17 comprisesamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:20.

In other aspects of this embodiment, a Clostridial botulinum serotype BHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQID NO: 20, at least 75% amino acid identity with amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 20, at least 80% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 20, at least 85% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 20,at least 90% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 20 or at least 95% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 20. In yet other aspects of this embodiment, a Clostridialbotulinum serotype B HA-17 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 20, at most 75% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 20,at most 80% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 20, at most 85% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 20, at most 90% amino acid identity with amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 20 or at most 95%amino acid identity with amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 20.

In other aspects of this embodiment, a Clostridial botulinum serotype BHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 20. In other aspects of this embodiment, aClostridial botulinum serotype B HA-17 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 20. In yet otheraspects of this embodiment, a Clostridial botulinum serotype B HA-17comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 20. In other aspects of this embodiment, a Clostridialbotulinum serotype B HA-17 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 9-50, amino acids 55-91, oramino acids 95-146 of SEQ ID NO: 20. In still other aspects of thisembodiment, a Clostridial botulinum serotype B HA-17 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 20.In other aspects of this embodiment, a Clostridial botulinum serotype BHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 20.

In other aspects of this embodiment, a Clostridial botulinum serotype BHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 20. In other aspects of this embodiment, aClostridial botulinum serotype B HA-17 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 20. In yet otheraspects of this embodiment, a Clostridial botulinum serotype B HA-17comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:20. In other aspects of this embodiment, a Clostridial botulinumserotype B HA-17 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 20. In still other aspects of thisembodiment, a Clostridial botulinum serotype B HA-17 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 20. Inother aspects of this embodiment, a Clostridial botulinum serotype BHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 20.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype B HA-17 comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype B HA-17 or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype B HA-17of SEQ ID NO: 20. In another aspect of this embodiment, a β-trefoildomain derived from a Clostridial botulinum serotype B HA-17 comprisesamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 20.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B HA-17 comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype B HA-17 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype B HA-17 of SEQ IDNO: 20. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB HA-17 comprises a modification of amino acids 51-54 or amino acids92-94 of SEQ ID NO: 20.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-17comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 20, at least75% amino acid identity with amino acids 51-54 or amino acids 92-94 ofSEQ ID NO: 20, at least 80% amino acid identity with amino acids 51-54or amino acids 92-94 of SEQ ID NO: 20, at least 85% amino acid identitywith amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 20, at least90% amino acid identity with amino acids 51-54 or amino acids 92-94 ofSEQ ID NO: 20 or at least 95% amino acid identity with amino acids 51-54or amino acids 92-94 of SEQ ID NO: 20. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype B HA-17 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 20, at most 75% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 20, at most 80%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 20, at most 85% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 20, at most 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 20 or at most 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 20.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-17comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 20. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-17 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 51-54 or amino acids 92-94 of SEQID NO: 20. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 20 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 51-54 or amino acids 92-94of SEQ ID NO: 20 can be replaced with phenylalanine. In yet otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype B HA-17 comprisesa polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 20. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-17 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 20. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype B HA-17 comprises a polypeptide having, e.g., at most one, two,three or four non-contiguous amino acid additions relative to aminoacids 51-54 or amino acids 92-94 of SEQ ID NO: 20. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B HA-17 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid additions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 20.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-17comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 20. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-17 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 51-54 or amino acids 92-94 of SEQID NO: 20. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 51-54 or amino acids 92-94of SEQ ID NO: 20 can be replaced with glycine. In other aspects of thisembodiment, contiguous amino acid substitutions of hydrophobic aminoacids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 20 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-17 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 20. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-17comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid deletions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 20. In still other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B HA-17 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid additionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 20. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B HA-17comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 20.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype C1 HA-17 comprises a β-trefoil domain derived from aClostridial botulinum serotype C1 HA-17 of SEQ ID NO: 21. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype C1 HA-17 comprises amino acids 9-146 of SEQ ID NO: 21. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype C1 HA-17 comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-17, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype C1 HA-17or a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype C1 HA-17 of SEQ ID NO: 21. In another aspect of thisembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype C1 HA-17 comprises amino acids 9-50, amino acids 55-91, oramino acids 95-146 of SEQ ID NO: 21.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-17 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-17 of SEQ ID NO: 21. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 HA-17 comprises amino acids9-146 of SEQ ID NO: 21. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-17 comprises a modified α-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype C1HA-17, a modified β-fold motif of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-17 or a modified γ-fold motif of a β-trefoildomain of a Clostridial botulinum serotype C1 HA-17 of SEQ ID NO: 21. Inanother aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-17comprises amino acids 9-50, amino acids 55-91, or amino acids 95-146 ofSEQ ID NO: 21.

In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQID NO: 21, at least 75% amino acid identity with amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 21, at least 80% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 21, at least 85% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21,at least 90% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 21 or at least 95% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 21. In yet other aspects of this embodiment, a Clostridialbotulinum serotype C1 HA-17 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 21, at most 75% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21,at most 80% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 21, at most 85% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 21, at most 90% amino acid identity with amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21 or at most 95%amino acid identity with amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 21.

In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 21. In other aspects of this embodiment, aClostridial botulinum serotype C1 HA-17 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In yet otheraspects of this embodiment, a Clostridial botulinum serotype C1 HA-17comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 21. In other aspects of this embodiment, a Clostridialbotulinum serotype C1 HA-17 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 9-50, amino acids 55-91, oramino acids 95-146 of SEQ ID NO: 21. In still other aspects of thisembodiment, a Clostridial botulinum serotype C1 HA-17 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21.In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 21.

In other aspects of this embodiment, a Clostridial botulinum serotype C1HA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 21. In other aspects of this embodiment, aClostridial botulinum serotype C1 HA-17 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 21. In yet otheraspects of this embodiment, a Clostridial botulinum serotype C1 HA-17comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:21. In other aspects of this embodiment, a Clostridial botulinumserotype C1 HA-17 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 21. In still other aspects of thisembodiment, a Clostridial botulinum serotype C1 HA-17 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 21. Inother aspects of this embodiment, a Clostridial botulinum serotype C1HA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 21.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype C1 HA-17 comprises a β4/β5 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-17 or a β8/β9hairpin turn of a β-trefoil domain of a Clostridial botulinum serotypeC1 HA-17 of SEQ ID NO: 21. In another aspect of this embodiment, aβ-trefoil domain derived from a Clostridial botulinum serotype C1 HA-17comprises amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-17 comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype C1 HA-17 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype C1 HA-17 of SEQ IDNO: 21. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 HA-17 comprises a modification of amino acids 51-54 or amino acids92-94 of SEQ ID NO: 21.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-17comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at least75% amino acid identity with amino acids 51-54 or amino acids 92-94 ofSEQ ID NO: 21, at least 80% amino acid identity with amino acids 51-54or amino acids 92-94 of SEQ ID NO: 21, at least 85% amino acid identitywith amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at least90% amino acid identity with amino acids 51-54 or amino acids 92-94 ofSEQ ID NO: 21 or at least 95% amino acid identity with amino acids 51-54or amino acids 92-94 of SEQ ID NO: 21. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 HA-17 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 21, at most 75% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21, at most 80%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 21, at most 85% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 21, at most 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 or at most 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 21.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-17comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-17 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 51-54 or amino acids 92-94 of SEQID NO: 21. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 21 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 51-54 or amino acids 92-94of SEQ ID NO: 21 can be replaced with phenylalanine. In yet otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype C1 HA-17comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-17 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 21. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype C1 HA-17 comprises a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid additions relative to aminoacids 51-54 or amino acids 92-94 of SEQ ID NO: 21. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 HA-17 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid additions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 21.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-17comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 21. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-17 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 51-54 or amino acids 92-94 of SEQID NO: 21. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 51-54 or amino acids 92-94of SEQ ID NO: 21 can be replaced with glycine. In other aspects of thisembodiment, contiguous amino acid substitutions of hydrophobic aminoacids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-17 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-17comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid deletions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 21. In still other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 HA-17 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid additionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 21. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 HA-17comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 21.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype D HA-17 comprises a β-trefoil domain derived from aClostridial botulinum serotype D HA-17 of SEQ ID NO: 22. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype D HA-17 comprises amino acids 9-146 of SEQ ID NO: 22. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype D HA-17 comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype D HA-17, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype D HA-17or a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype D HA-17 of SEQ ID NO: 22. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype D HA-17comprises amino acids 9-50, amino acids 55-91, or amino acids 95-146 ofSEQ ID NO: 22.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype D HA-17 comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-17 of SEQ ID NO: 22. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype D HA-17 comprises amino acids9-146 of SEQ ID NO: 22. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-17 comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype D HA-17, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype D HA-17 or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype D HA-17 of SEQ ID NO: 22. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype D HA-17 comprisesamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:22.

In other aspects of this embodiment, a Clostridial botulinum serotype DHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQID NO: 22, at least 75% amino acid identity with amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 22, at least 80% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 22, at least 85% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22,at least 90% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 22 or at least 95% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 22. In yet other aspects of this embodiment, a Clostridialbotulinum serotype D HA-17 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most 70% aminoacid identity with amino acids 9-50, amino acids 55-91, or amino acids95-146 of SEQ ID NO: 22, at most 75% amino acid identity with aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22,at most 80% amino acid identity with amino acids 9-50, amino acids55-91, or amino acids 95-146 of SEQ ID NO: 22, at most 85% amino acididentity with amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 22, at most 90% amino acid identity with amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22 or at most 95%amino acid identity with amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 22.

In other aspects of this embodiment, a Clostridial botulinum serotype DHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 22. In other aspects of this embodiment, aClostridial botulinum serotype D HA-17 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 9-50,amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In yet otheraspects of this embodiment, a Clostridial botulinum serotype D HA-17comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 22. In other aspects of this embodiment, a Clostridialbotulinum serotype D HA-17 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 9-50, amino acids 55-91, oramino acids 95-146 of SEQ ID NO: 22. In still other aspects of thisembodiment, a Clostridial botulinum serotype D HA-17 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22.In other aspects of this embodiment, a Clostridial botulinum serotype DHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 22.

In other aspects of this embodiment, a Clostridial botulinum serotype DHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 22. In other aspects of this embodiment, aClostridial botulinum serotype D HA-17 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 9-50, aminoacids 55-91, or amino acids 95-146 of SEQ ID NO: 22. In yet otheraspects of this embodiment, a Clostridial botulinum serotype D HA-17comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10 or 20 contiguous amino acid deletions relative toamino acids 9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO:22. In other aspects of this embodiment, a Clostridial botulinumserotype D HA-17 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino aciddeletions relative to amino acids 9-50, amino acids 55-91, or aminoacids 95-146 of SEQ ID NO: 22. In still other aspects of thisembodiment, a Clostridial botulinum serotype D HA-17 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids9-50, amino acids 55-91, or amino acids 95-146 of SEQ ID NO: 22. Inother aspects of this embodiment, a Clostridial botulinum serotype DHA-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 9-50, amino acids 55-91, or amino acids 95-146of SEQ ID NO: 22.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype D HA-17 comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype D HA-17 or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype D HA-17of SEQ ID NO: 22. In another aspect of this embodiment, a β-trefoildomain derived from a Clostridial botulinum serotype D HA-17 comprisesamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype D HA-17 comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype D HA-17 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype D HA-17 of SEQ IDNO: 22. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeD HA-17 comprises a modification of amino acids 51-54 or amino acids92-94 of SEQ ID NO: 22.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D HA-17comprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at least75% amino acid identity with amino acids 51-54 or amino acids 92-94 ofSEQ ID NO: 22, at least 80% amino acid identity with amino acids 51-54or amino acids 92-94 of SEQ ID NO: 22, at least 85% amino acid identitywith amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at least90% amino acid identity with amino acids 51-54 or amino acids 92-94 ofSEQ ID NO: 22 or at least 95% amino acid identity with amino acids 51-54or amino acids 92-94 of SEQ ID NO: 22. In yet other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype D HA-17 comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 22, at most 75% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22, at most 80%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 22, at most 85% amino acid identity with amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 22, at most 90% amino acid identity withamino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 or at most 95%amino acid identity with amino acids 51-54 or amino acids 92-94 of SEQID NO: 22.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D HA-17comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-17 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 51-54 or amino acids 92-94 of SEQID NO: 22. In other aspects of this embodiment, a non-contiguous aminoacid substitution of any amino acid from amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 22 can be replaced with glycine. In otheraspects of this embodiment, a non-contiguous amino acid substitution ofany hydrophobic amino acid from amino acids 51-54 or amino acids 92-94of SEQ ID NO: 22 can be replaced with phenylalanine. In yet otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype D HA-17 comprisesa polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-17 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino aciddeletions relative to amino acids 51-54 or amino acids 92-94 of SEQ IDNO: 22. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype D HA-17 comprises a polypeptide having, e.g., at most one, two,three or four non-contiguous amino acid additions relative to aminoacids 51-54 or amino acids 92-94 of SEQ ID NO: 22. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype D HA-17 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid additions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 22.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D HA-17comprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 51-54 oramino acids 92-94 of SEQ ID NO: 22. In other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-17 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 51-54 or amino acids 92-94 of SEQID NO: 22. In other aspects of this embodiment, contiguous amino acidsubstitutions of amino acids from amino acids 51-54 or amino acids 92-94of SEQ ID NO: 22 can be replaced with glycine. In other aspects of thisembodiment, contiguous amino acid substitutions of hydrophobic aminoacids from amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-17 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D HA-17comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid deletions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 22. In still other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D HA-17 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid additionsrelative to amino acids 51-54 or amino acids 92-94 of SEQ ID NO: 22. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D HA-17comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 51-54 or aminoacids 92-94 of SEQ ID NO: 22.

In an embodiment, a modified Clostridial toxin disclosed in the presentspecification comprises an enhanced targeting domain comprising aβ-trefoil domain derived from a Clostridial NTNH. In an aspect of thisembodiment, a β-trefoil domain derived from a Clostridial NTNHcomprises, e.g., a β-trefoil domain derived from a Clostridial botulinumserotype A NTNH, a β-trefoil domain derived from a Clostridial botulinumserotype B NTNH, a β-trefoil domain derived from a Clostridial botulinumserotype C1 NTNH, a β-trefoil domain derived from a Clostridialbotulinum serotype D NTNH, a β-trefoil domain derived from a Clostridialbotulinum serotype E NTNH, a β-trefoil domain derived from a Clostridialbotulinum serotype F NTNH or a β-trefoil domain derived from aClostridial botulinum serotype G NTNH. In another aspect of thisembodiment, a β-trefoil domain derived from a Clostridial NTNH comprisesa α-fold motif of a 8-trefoil domain of a Clostridial NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial NTNH or a γ-fold motif of aβ-trefoil domain of a Clostridial NTNH.

In an embodiment, a modified Clostridial toxin disclosed in the presentspecification comprises an enhanced targeting domain comprising aβ-trefoil domain with enhanced binding activity derived from aClostridial NTNH. In an aspect of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial NTNHcomprises, e.g., a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A NTNH, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype B NTNH, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 NTNH, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype D NTNH, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype E NTNH, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype F NTNH or a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeG NTNH. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial NTNH comprises amodified α-fold motif of a β-trefoil domain of a Clostridial NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial NTNH or amodified γ-fold motif of a β-trefoil domain of a Clostridial NTNH.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A NTNH comprises a β-trefoil domain derived from aClostridial botulinum serotype A NTNH of SEQ ID NO: 23. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype A NTNH comprises amino acids 1050-1194 of SEQ ID NO: 23. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype A NTNH comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype A NTNHor a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A NTNH of SEQ ID NO: 23. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype A NTNHcomprises amino acids 1050-1097, amino acids 1111-1138, or amino acids1149-1194 of SEQ ID NO: 23.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A NTNH comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A NTNH of SEQ ID NO: 23. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises amino acids1050-1194 of SEQ ID NO: 23. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A NTNH comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype A NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A NTNH or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype A NTNH of SEQ ID NO: 23. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype A NTNH comprisesamino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194of SEQ ID NO: 23.

In other aspects of this embodiment, a Clostridial botulinum serotype ANTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1050-1097, amino acids 1111-1138, or amino acids1149-1194 of SEQ ID NO: 23, at least 75% amino acid identity with aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 23, at least 80% amino acid identity with amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 23, atleast 85% amino acid identity with amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 23, at least 90% aminoacid identity with amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 23 or at least 95% amino acididentity with amino acids 1050-1097, amino acids 1111-1138, or aminoacids 1149-1194 of SEQ ID NO: 23. In yet other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:23, at most 75% amino acid identity with amino acids 1050-1097, aminoacids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 23, at most 80%amino acid identity with amino acids 1050-1097, amino acids 1111-1138,or amino acids 1149-1194 of SEQ ID NO: 23, at most 85% amino acididentity with amino acids 1050-1097, amino acids 1111-1138, or aminoacids 1149-1194 of SEQ ID NO: 23, at most 90% amino acid identity withamino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194of SEQ ID NO: 23 or at most 95% amino acid identity with amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:23.

In other aspects of this embodiment, a Clostridial botulinum serotype ANTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1050-1097, amino acids 1111-1138,or amino acids 1149-1194 of SEQ ID NO: 23. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 23. In yet other aspects of this embodiment, a Clostridialbotulinum serotype A NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 23. In other aspectsof this embodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 23. In still other aspects of this embodiment, a Clostridialbotulinum serotype A NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 23. In other aspectsof this embodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 23.

In other aspects of this embodiment, a Clostridial botulinum serotype ANTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1050-1097, amino acids 1111-1138,or amino acids 1149-1194 of SEQ ID NO: 23. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 23. In yet other aspects of this embodiment, a Clostridialbotulinum serotype A NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 1050-1097, amino acids 1111-1138,or amino acids 1149-1194 of SEQ ID NO: 23. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:23. In still other aspects of this embodiment, a Clostridial botulinumserotype A NTNH comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 23. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:23.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A NTNH comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype A NTNH or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype A NTNH ofSEQ ID NO: 23. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype A NTNH comprises aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A NTNH comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A NTNH or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A NTNH of SEQ IDNO: 23. In another aspect of this embodiment, a 11-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA NTNH comprises a modification of amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 23.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23, atleast 75% amino acid identity with amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 23, at least 80% amino acid identity with aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23, at least 85%amino acid identity with amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 23, at least 90% amino acid identity with amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23 or at least 95%amino acid identity with amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 23. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A NTNH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 23, at most 75% amino acid identity with aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23, at most 80%amino acid identity with amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 23, at most 85% amino acid identity with amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23, at most 90% aminoacid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQID NO: 23 or at most 95% amino acid identity with amino acids 1098-1110or amino acids 1139-1148 of SEQ ID NO: 23.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A NTNH comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO:23 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A NTNH comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 23. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype A NTNH comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23. In stillother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 1098-1110 oramino acids 1139-1148 of SEQ ID NO: 23. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 23.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 1098-1110 oramino acids 1139-1148 of SEQ ID NO: 23. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 23. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1098-1110 or aminoacids 1139-1148 of SEQ ID NO: 23 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 23 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 1098-1110 oramino acids 1139-1148 of SEQ ID NO: 23. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 23. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A NTNH comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 23. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 1098-1110 oramino acids 1139-1148 of SEQ ID NO: 23.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A NTNH comprises a β-trefoil domain derived from aClostridial botulinum serotype A NTNH of SEQ ID NO: 24. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype A NTNH comprises amino acids 1050-1194 of SEQ ID NO: 24. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype A NTNH comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype A NTNHor a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A NTNH of SEQ ID NO: 24. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype A NTNHcomprises amino acids 1050-1097, amino acids 1111-1139, or amino acids1149-1199 of SEQ ID NO: 24.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A NTNH comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A NTNH of SEQ ID NO: 24. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises amino acids1050-1194 of SEQ ID NO: 24. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A NTNH comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype A NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A NTNH or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype A NTNH of SEQ ID NO: 24. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype A NTNH comprisesamino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199of SEQ ID NO: 24.

In other aspects of this embodiment, a Clostridial botulinum serotype ANTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1050-1097, amino acids 1111-1139, or amino acids1149-1199 of SEQ ID NO: 24, at least 75% amino acid identity with aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQID NO: 24, at least 80% amino acid identity with amino acids 1050-1097,amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 24, atleast 85% amino acid identity with amino acids 1050-1097, amino acids1111-1139, or amino acids 1149-1199 of SEQ ID NO: 24, at least 90% aminoacid identity with amino acids 1050-1097, amino acids 1111-1139, oramino acids 1149-1199 of SEQ ID NO: 24 or at least 95% amino acididentity with amino acids 1050-1097, amino acids 1111-1139, or aminoacids 1149-1199 of SEQ ID NO: 24. In yet other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO:24, at most 75% amino acid identity with amino acids 1050-1097, aminoacids 1111-1139, or amino acids 1149-1199 of SEQ ID NO: 24, at most 80%amino acid identity with amino acids 1050-1097, amino acids 1111-1139,or amino acids 1149-1199 of SEQ ID NO: 24, at most 85% amino acididentity with amino acids 1050-1097, amino acids 1111-1139, or aminoacids 1149-1199 of SEQ ID NO: 24, at most 90% amino acid identity withamino acids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199of SEQ ID NO: 24 or at most 95% amino acid identity with amino acids1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO:24.

In other aspects of this embodiment, a Clostridial botulinum serotype ANTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1050-1097, amino acids 1111-1139,or amino acids 1149-1199 of SEQ ID NO: 24. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQID NO: 24. In yet other aspects of this embodiment, a Clostridialbotulinum serotype A NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 1050-1097, amino acids1111-1139, or amino acids 1149-1199 of SEQ ID NO: 24. In other aspectsof this embodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQID NO: 24. In still other aspects of this embodiment, a Clostridialbotulinum serotype A NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1050-1097, amino acids1111-1139, or amino acids 1149-1199 of SEQ ID NO: 24. In other aspectsof this embodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQID NO: 24.

In other aspects of this embodiment, a Clostridial botulinum serotype ANTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1050-1097, amino acids 1111-1139,or amino acids 1149-1199 of SEQ ID NO: 24. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQID NO: 24. In yet other aspects of this embodiment, a Clostridialbotulinum serotype A NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 1050-1097, amino acids 1111-1139,or amino acids 1149-1199 of SEQ ID NO: 24. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO:24. In still other aspects of this embodiment, a Clostridial botulinumserotype A NTNH comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 1050-1097, amino acids 1111-1139, oramino acids 1149-1199 of SEQ ID NO: 24. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids1050-1097, amino acids 1111-1139, or amino acids 1149-1199 of SEQ ID NO:24.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A NTNH comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype A NTNH or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype A NTNH ofSEQ ID NO: 24. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype A NTNH comprises aminoacids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A NTNH comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A NTNH or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A NTNH of SEQ IDNO: 24. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA NTNH comprises a modification of amino acids 1098-1110 or amino acids1140-1148 of SEQ ID NO: 24.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24, atleast 75% amino acid identity with amino acids 1098-1110 or amino acids1140-1148 of SEQ ID NO: 24, at least 80% amino acid identity with aminoacids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24, at least 85%amino acid identity with amino acids 1098-1110 or amino acids 1140-1148of SEQ ID NO: 24, at least 90% amino acid identity with amino acids1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24 or at least 95%amino acid identity with amino acids 1098-1110 or amino acids 1140-1148of SEQ ID NO: 24. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A NTNH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1098-1110 or amino acids1140-1148 of SEQ ID NO: 24, at most 75% amino acid identity with aminoacids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24, at most 80%amino acid identity with amino acids 1098-1110 or amino acids 1140-1148of SEQ ID NO: 24, at most 85% amino acid identity with amino acids1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24, at most 90% aminoacid identity with amino acids 1098-1110 or amino acids 1140-1148 of SEQID NO: 24 or at most 95% amino acid identity with amino acids 1098-1110or amino acids 1140-1148 of SEQ ID NO: 24.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A NTNH comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO:24 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A NTNH comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1140-1148 ofSEQ ID NO: 24. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype A NTNH comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24. In stillother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 1098-1110 oramino acids 1140-1148 of SEQ ID NO: 24. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1140-1148 ofSEQ ID NO: 24.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 1098-1110 oramino acids 1140-1148 of SEQ ID NO: 24. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 1098-1110 or amino acids 1140-1148of SEQ ID NO: 24. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1098-1110 or aminoacids 1140-1148 of SEQ ID NO: 24 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1098-1110 or amino acids1140-1148 of SEQ ID NO: 24 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 1098-1110 oramino acids 1140-1148 of SEQ ID NO: 24. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1140-1148 ofSEQ ID NO: 24. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A NTNH comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 1098-1110 or amino acids 1140-1148 of SEQ ID NO: 24. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 1098-1110 oramino acids 1140-1148 of SEQ ID NO: 24.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A NTNH comprises a β-trefoil domain derived from aClostridial botulinum serotype A NTNH of SEQ ID NO: 25. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype A NTNH comprises amino acids 1050-1194 of SEQ ID NO: 25. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype A NTNH comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype A NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype A NTNHor a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A NTNH of SEQ ID NO: 25. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype A NTNHcomprises amino acids 1050-1097, amino acids 1111-1138, or amino acids1149-1194 of SEQ ID NO: 25.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A NTNH comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A NTNH of SEQ ID NO: 25. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises amino acids1050-1194 of SEQ ID NO: 25. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A NTNH comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype A NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype A NTNH or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype A NTNH of SEQ ID NO: 25. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype A NTNH comprisesamino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194of SEQ ID NO: 25.

In other aspects of this embodiment, a Clostridial botulinum serotype ANTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1050-1097, amino acids 1111-1138, or amino acids1149-1194 of SEQ ID NO: 25, at least 75% amino acid identity with aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 25, at least 80% amino acid identity with amino acids 1050-1097,amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, atleast 85% amino acid identity with amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at least 90% aminoacid identity with amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 25 or at least 95% amino acididentity with amino acids 1050-1097, amino acids 1111-1138, or aminoacids 1149-1194 of SEQ ID NO: 25. In yet other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:25, at most 75% amino acid identity with amino acids 1050-1097, aminoacids 1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25, at most 80%amino acid identity with amino acids 1050-1097, amino acids 1111-1138,or amino acids 1149-1194 of SEQ ID NO: 25, at most 85% amino acididentity with amino acids 1050-1097, amino acids 1111-1138, or aminoacids 1149-1194 of SEQ ID NO: 25, at most 90% amino acid identity withamino acids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194of SEQ ID NO: 25 or at most 95% amino acid identity with amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:25.

In other aspects of this embodiment, a Clostridial botulinum serotype ANTNH comprising a 11-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1050-1097, amino acids 1111-1138,or amino acids 1149-1194 of SEQ ID NO: 25. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 25. In yet other aspects of this embodiment, a Clostridialbotulinum serotype A NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In other aspectsof this embodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 25. In still other aspects of this embodiment, a Clostridialbotulinum serotype A NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1050-1097, amino acids1111-1138, or amino acids 1149-1194 of SEQ ID NO: 25. In other aspectsof this embodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 25.

In other aspects of this embodiment, a Clostridial botulinum serotype ANTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1050-1097, amino acids 1111-1138,or amino acids 1149-1194 of SEQ ID NO: 25. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQID NO: 25. In yet other aspects of this embodiment, a Clostridialbotulinum serotype A NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 1050-1097, amino acids 1111-1138,or amino acids 1149-1194 of SEQ ID NO: 25. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:25. In still other aspects of this embodiment, a Clostridial botulinumserotype A NTNH comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 1050-1097, amino acids 1111-1138, oramino acids 1149-1194 of SEQ ID NO: 25. In other aspects of thisembodiment, a Clostridial botulinum serotype A NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids1050-1097, amino acids 1111-1138, or amino acids 1149-1194 of SEQ ID NO:25.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype A NTNH comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype A NTNH or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype A NTNH ofSEQ ID NO: 25. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype A NTNH comprises aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A NTNH comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype A NTNH or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype A NTNH of SEQ IDNO: 25. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeA NTNH comprises a modification of amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 25.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, atleast 75% amino acid identity with amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 25, at least 80% amino acid identity with aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at least 85%amino acid identity with amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 25, at least 90% amino acid identity with amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25 or at least 95%amino acid identity with amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 25. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A NTNH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 25, at most 75% amino acid identity with aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at most 80%amino acid identity with amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 25, at most 85% amino acid identity with amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25, at most 90% aminoacid identity with amino acids 1098-1110 or amino acids 1139-1148 of SEQID NO: 25 or at most 95% amino acid identity with amino acids 1098-1110or amino acids 1139-1148 of SEQ ID NO: 25.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype A NTNH comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO:25 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype A NTNH comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 25. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype A NTNH comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. In stillother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 1098-1110 oramino acids 1139-1148 of SEQ ID NO: 25. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 25.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 1098-1110 oramino acids 1139-1148 of SEQ ID NO: 25. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 1098-1110 or amino acids 1139-1148of SEQ ID NO: 25. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1098-1110 or aminoacids 1139-1148 of SEQ ID NO: 25 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1098-1110 or amino acids1139-1148 of SEQ ID NO: 25 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 1098-1110 oramino acids 1139-1148 of SEQ ID NO: 25. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype A NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1139-1148 ofSEQ ID NO: 25. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype A NTNH comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 1098-1110 or amino acids 1139-1148 of SEQ ID NO: 25. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype A NTNHcomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 1098-1110 oramino acids 1139-1148 of SEQ ID NO: 25.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype B NTNH comprises a β-trefoil domain derived from aClostridial botulinum serotype B NTNH of SEQ ID NO: 26. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype B NTNH comprises amino acids 1049-1198 of SEQ ID NO: 26. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype B NTNH comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype B NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype B NTNHor a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype B NTNH of SEQ ID NO: 26. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype B NTNHcomprises amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1198 of SEQ ID NO: 26.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B NTNH comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B NTNH of SEQ ID NO: 26. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype B NTNH comprises amino acids1049-1198 of SEQ ID NO: 26. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B NTNH comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype B NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype B NTNH or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype B NTNH of SEQ ID NO: 26. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype B NTNH comprisesamino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198of SEQ ID NO: 26.

In other aspects of this embodiment, a Clostridial botulinum serotype BNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1198 of SEQ ID NO: 26, at least 75% amino acid identity with aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQID NO: 26, at least 80% amino acid identity with amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 26, atleast 85% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1198 of SEQ ID NO: 26, at least 90% aminoacid identity with amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1198 of SEQ ID NO: 26 or at least 95% amino acididentity with amino acids 1049-1096, amino acids 1110-1138, or aminoacids 1148-1198 of SEQ ID NO: 26. In yet other aspects of thisembodiment, a Clostridial botulinum serotype B NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO:26, at most 75% amino acid identity with amino acids 1049-1096, aminoacids 1110-1138, or amino acids 1148-1198 of SEQ ID NO: 26, at most 80%amino acid identity with amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1198 of SEQ ID NO: 26, at most 85% amino acididentity with amino acids 1049-1096, amino acids 1110-1138, or aminoacids 1148-1198 of SEQ ID NO: 26, at most 90% amino acid identity withamino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198of SEQ ID NO: 26 or at most 95% amino acid identity with amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO:26.

In other aspects of this embodiment, a Clostridial botulinum serotype BNTNH comprising a 11-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1198 of SEQ ID NO: 26. In other aspects of thisembodiment, a Clostridial botulinum serotype B NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQID NO: 26. In yet other aspects of this embodiment, a Clostridialbotulinum serotype B NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1198 of SEQ ID NO: 26. In other aspectsof this embodiment, a Clostridial botulinum serotype B NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQID NO: 26. In still other aspects of this embodiment, a Clostridialbotulinum serotype B NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1198 of SEQ ID NO: 26. In other aspectsof this embodiment, a Clostridial botulinum serotype B NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQID NO: 26.

In other aspects of this embodiment, a Clostridial botulinum serotype BNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1198 of SEQ ID NO: 26. In other aspects of thisembodiment, a Clostridial botulinum serotype B NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQID NO: 26. In yet other aspects of this embodiment, a Clostridialbotulinum serotype B NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1198 of SEQ ID NO: 26. In other aspects of thisembodiment, a Clostridial botulinum serotype B NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO:26. In still other aspects of this embodiment, a Clostridial botulinumserotype B NTNH comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1198 of SEQ ID NO: 26. In other aspects of thisembodiment, a Clostridial botulinum serotype B NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1198 of SEQ ID NO:26.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype B NTNH comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype B NTNH or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype B NTNH ofSEQ ID NO: 26. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype B NTNH comprises aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B NTNH comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype B NTNH or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype B NTNH of SEQ IDNO: 26. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeB NTNH comprises a modification of amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 26.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B NTNHcomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26, atleast 75% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 26, at least 80% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26, at least 85%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 26, at least 90% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26 or at least 95%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 26. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype B NTNH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 26, at most 75% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26, at most 80%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 26, at most 85% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26, at most 90% aminoacid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQID NO: 26 or at most 95% amino acid identity with amino acids 1097-1109or amino acids 1139-1147 of SEQ ID NO: 26.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype B NTNH comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:26 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype B NTNH comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 26. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype B NTNH comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26. In stillother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 26. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype B NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 26.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 26. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype B NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 26. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1097-1109 or aminoacids 1139-1147 of SEQ ID NO: 26 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 26 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 26. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype B NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 26. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype B NTNH comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 26. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype B NTNHcomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 26.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype C1 NTNH comprises a β-trefoil domain derived from aClostridial botulinum serotype C1 NTNH of SEQ ID NO: 27. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype C1 NTNH comprises amino acids 1049-1197 of SEQ ID NO: 27. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype C1 NTNH comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype C1 NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype C1 NTNHor a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype C1 NTNH of SEQ ID NO: 27. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype C1 NTNHcomprises amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 27.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 NTNH comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 NTNH of SEQ ID NO: 27. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 NTNH comprises amino acids1049-1197 of SEQ ID NO: 27. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 NTNH comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype C1 NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype C1 NTNH or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype C1 NTNH of SEQ ID NO: 27. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype C1 NTNH comprisesamino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197of SEQ ID NO: 27.

In other aspects of this embodiment, a Clostridial botulinum serotype C1NTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 27, at least 75% amino acid identity with aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 27, at least 80% amino acid identity with amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 27, atleast 85% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 27, at least 90% aminoacid identity with amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 27 or at least 95% amino acididentity with amino acids 1049-1096, amino acids 1110-1138, or aminoacids 1148-1197 of SEQ ID NO: 27. In yet other aspects of thisembodiment, a Clostridial botulinum serotype C1 NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:27, at most 75% amino acid identity with amino acids 1049-1096, aminoacids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 27, at most 80%amino acid identity with amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1197 of SEQ ID NO: 27, at most 85% amino acididentity with amino acids 1049-1096, amino acids 1110-1138, or aminoacids 1148-1197 of SEQ ID NO: 27, at most 90% amino acid identity withamino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197of SEQ ID NO: 27 or at most 95% amino acid identity with amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:27.

In other aspects of this embodiment, a Clostridial botulinum serotype C1NTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1197 of SEQ ID NO: 27. In other aspects of thisembodiment, a Clostridial botulinum serotype C1 NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 27. In yet other aspects of this embodiment, a Clostridialbotulinum serotype C1 NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 27. In other aspectsof this embodiment, a Clostridial botulinum serotype C1 NTNH comprisinga β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine, 10 or 20 non-contiguous amino acid deletionsrelative to amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 27. In still other aspects of this embodiment, aClostridial botulinum serotype C1 NTNH comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 27. Inother aspects of this embodiment, a Clostridial botulinum serotype C1NTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 27.

In other aspects of this embodiment, a Clostridial botulinum serotype C1NTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1197 of SEQ ID NO: 27. In other aspects of thisembodiment, a Clostridial botulinum serotype C1 NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 27. In yet other aspects of this embodiment, a Clostridialbotulinum serotype C1 NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1197 of SEQ ID NO: 27. In other aspects of thisembodiment, a Clostridial botulinum serotype C1 NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:27. In still other aspects of this embodiment, a Clostridial botulinumserotype C1 NTNH comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 27. In other aspects of thisembodiment, a Clostridial botulinum serotype C1 NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:27.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype C1 NTNH comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype C1 NTNH or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype C1 NTNHof SEQ ID NO: 27. In another aspect of this embodiment, a β-trefoildomain derived from a Clostridial botulinum serotype C1 NTNH comprisesamino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 NTNH comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype C1 NTNH or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype C1 NTNH of SEQ IDNO: 27. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeC1 NTNH comprises a modification of amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 27.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 NTNHcomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27, atleast 75% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 27, at least 80% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27, at least 85%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 27, at least 90% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27 or at least 95%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 27. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype C1 NTNH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 27, at most 75% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27, at most 80%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 27, at most 85% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27, at most 90% aminoacid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQID NO: 27 or at most 95% amino acid identity with amino acids 1097-1109or amino acids 1139-1147 of SEQ ID NO: 27.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype C1 NTNH comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:27 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype C1 NTNH comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 27. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype C1 NTNH comprises a polypeptide having, e.g., at least one,two, three or four non-contiguous amino acid deletions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27. In stillother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 27. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 27.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 27. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 27. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1097-1109 or aminoacids 1139-1147 of SEQ ID NO: 27 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 27 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 27. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype C1 NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 27. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype C1 NTNH comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 27. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype C1 NTNHcomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 27.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype D NTNH comprises a β-trefoil domain derived from aClostridial botulinum serotype D NTNH of SEQ ID NO: 28. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype D NTNH comprises amino acids 1049-1197 of SEQ ID NO: 28. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype D NTNH comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype D NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype D NTNHor a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype D NTNH of SEQ ID NO: 28. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype D NTNHcomprises amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 28.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype D NTNH comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D NTNH of SEQ ID NO: 28. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype D NTNH comprises amino acids1049-1197 of SEQ ID NO: 28. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D NTNH comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype D NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype D NTNH or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype D NTNH of SEQ ID NO: 28. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype D NTNH comprisesamino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197of SEQ ID NO: 28.

In other aspects of this embodiment, a Clostridial botulinum serotype DNTNH comprising a 11-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1049-1096, amino acids 1110-1138, or amino acids1148-1197 of SEQ ID NO: 28, at least 75% amino acid identity with aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 28, at least 80% amino acid identity with amino acids 1049-1096,amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 28, atleast 85% amino acid identity with amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 28, at least 90% aminoacid identity with amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 28 or at least 95% amino acididentity with amino acids 1049-1096, amino acids 1110-1138, or aminoacids 1148-1197 of SEQ ID NO: 28. In yet other aspects of thisembodiment, a Clostridial botulinum serotype D NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:28, at most 75% amino acid identity with amino acids 1049-1096, aminoacids 1110-1138, or amino acids 1148-1197 of SEQ ID NO: 28, at most 80%amino acid identity with amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1197 of SEQ ID NO: 28, at most 85% amino acididentity with amino acids 1049-1096, amino acids 1110-1138, or aminoacids 1148-1197 of SEQ ID NO: 28, at most 90% amino acid identity withamino acids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197of SEQ ID NO: 28 or at most 95% amino acid identity with amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:28.

In other aspects of this embodiment, a Clostridial botulinum serotype DNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1197 of SEQ ID NO: 28. In other aspects of thisembodiment, a Clostridial botulinum serotype D NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 28. In yet other aspects of this embodiment, a Clostridialbotulinum serotype D NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 28. In other aspectsof this embodiment, a Clostridial botulinum serotype D NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 28. In still other aspects of this embodiment, a Clostridialbotulinum serotype D NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1049-1096, amino acids1110-1138, or amino acids 1148-1197 of SEQ ID NO: 28. In other aspectsof this embodiment, a Clostridial botulinum serotype D NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 28.

In other aspects of this embodiment, a Clostridial botulinum serotype DNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1197 of SEQ ID NO: 28. In other aspects of thisembodiment, a Clostridial botulinum serotype D NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQID NO: 28. In yet other aspects of this embodiment, a Clostridialbotulinum serotype D NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 1049-1096, amino acids 1110-1138,or amino acids 1148-1197 of SEQ ID NO: 28. In other aspects of thisembodiment, a Clostridial botulinum serotype D NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:28. In still other aspects of this embodiment, a Clostridial botulinumserotype D NTNH comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 1049-1096, amino acids 1110-1138, oramino acids 1148-1197 of SEQ ID NO: 28. In other aspects of thisembodiment, a Clostridial botulinum serotype D NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids1049-1096, amino acids 1110-1138, or amino acids 1148-1197 of SEQ ID NO:28.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype D NTNH comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype D NTNH or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype D NTNH ofSEQ ID NO: 28. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype D NTNH comprises aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype D NTNH comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype D NTNH or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype D NTNH of SEQ IDNO: 28. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeD NTNH comprises a modification of amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 28.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D NTNHcomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, atleast 75% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 28, at least 80% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at least 85%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 28, at least 90% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28 or at least 95%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 28. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype D NTNH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 28, at most 75% amino acid identity with aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at most 80%amino acid identity with amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 28, at most 85% amino acid identity with amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28, at most 90% aminoacid identity with amino acids 1097-1109 or amino acids 1139-1147 of SEQID NO: 28 or at most 95% amino acid identity with amino acids 1097-1109or amino acids 1139-1147 of SEQ ID NO: 28.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype D NTNH comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO:28 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype D NTNH comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 28. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype D NTNH comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. In stillother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 28. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype D NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 28.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 28. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype D NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 1097-1109 or amino acids 1139-1147of SEQ ID NO: 28. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1097-1109 or aminoacids 1139-1147 of SEQ ID NO: 28 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1097-1109 or amino acids1139-1147 of SEQ ID NO: 28 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 28. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype D NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1097-1109 or amino acids 1139-1147 ofSEQ ID NO: 28. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype D NTNH comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 1097-1109 or amino acids 1139-1147 of SEQ ID NO: 28. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype D NTNHcomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 1097-1109 oramino acids 1139-1147 of SEQ ID NO: 28.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype E NTNH comprises a β-trefoil domain derived from aClostridial botulinum serotype E NTNH of SEQ ID NO: 29. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype E NTNH comprises amino acids 1014-1163 of SEQ ID NO: 29. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype E NTNH comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype E NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype E NTNHor a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype E NTNH of SEQ ID NO: 29. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype E NTNHcomprises amino acids 1014-1061, amino acids 1075-1103, or amino acids1114-1163 of SEQ ID NO: 29.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype E NTNH comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype E NTNH of SEQ ID NO: 29. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype E NTNH comprises amino acids1014-1163 of SEQ ID NO: 29. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype E NTNH comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype E NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype E NTNH or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype E NTNH of SEQ ID NO: 29. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype E NTNH comprisesamino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163of SEQ ID NO: 29.

In other aspects of this embodiment, a Clostridial botulinum serotype ENTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1014-1061, amino acids 1075-1103, or amino acids1114-1163 of SEQ ID NO: 29, at least 75% amino acid identity with aminoacids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQID NO: 29, at least 80% amino acid identity with amino acids 1014-1061,amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 29, atleast 85% amino acid identity with amino acids 1014-1061, amino acids1075-1103, or amino acids 1114-1163 of SEQ ID NO: 29, at least 90% aminoacid identity with amino acids 1014-1061, amino acids 1075-1103, oramino acids 1114-1163 of SEQ ID NO: 29 or at least 95% amino acididentity with amino acids 1014-1061, amino acids 1075-1103, or aminoacids 1114-1163 of SEQ ID NO: 29. In yet other aspects of thisembodiment, a Clostridial botulinum serotype E NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO:29, at most 75% amino acid identity with amino acids 1014-1061, aminoacids 1075-1103, or amino acids 1114-1163 of SEQ ID NO: 29, at most 80%amino acid identity with amino acids 1014-1061, amino acids 1075-1103,or amino acids 1114-1163 of SEQ ID NO: 29, at most 85% amino acididentity with amino acids 1014-1061, amino acids 1075-1103, or aminoacids 1114-1163 of SEQ ID NO: 29, at most 90% amino acid identity withamino acids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163of SEQ ID NO: 29 or at most 95% amino acid identity with amino acids1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO:29.

In other aspects of this embodiment, a Clostridial botulinum serotype ENTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1014-1061, amino acids 1075-1103,or amino acids 1114-1163 of SEQ ID NO: 29. In other aspects of thisembodiment, a Clostridial botulinum serotype E NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQID NO: 29. In yet other aspects of this embodiment, a Clostridialbotulinum serotype E NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 1014-1061, amino acids1075-1103, or amino acids 1114-1163 of SEQ ID NO: 29. In other aspectsof this embodiment, a Clostridial botulinum serotype E NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQID NO: 29. In still other aspects of this embodiment, a Clostridialbotulinum serotype E NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1014-1061, amino acids1075-1103, or amino acids 1114-1163 of SEQ ID NO: 29. In other aspectsof this embodiment, a Clostridial botulinum serotype E NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQID NO: 29.

In other aspects of this embodiment, a Clostridial botulinum serotype ENTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1014-1061, amino acids 1075-1103,or amino acids 1114-1163 of SEQ ID NO: 29. In other aspects of thisembodiment, a Clostridial botulinum serotype E NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQID NO: 29. In yet other aspects of this embodiment, a Clostridialbotulinum serotype E NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 1014-1061, amino acids 1075-1103,or amino acids 1114-1163 of SEQ ID NO: 29. In other aspects of thisembodiment, a Clostridial botulinum serotype E NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO:29. In still other aspects of this embodiment, a Clostridial botulinumserotype E NTNH comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 1014-1061, amino acids 1075-1103, oramino acids 1114-1163 of SEQ ID NO: 29. In other aspects of thisembodiment, a Clostridial botulinum serotype E NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids1014-1061, amino acids 1075-1103, or amino acids 1114-1163 of SEQ ID NO:29.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype E NTNH comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype E NTNH or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype E NTNH ofSEQ ID NO: 29. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype E NTNH comprises aminoacids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype E NTNH comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype E NTNH or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype E NTNH of SEQ IDNO: 29. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeE NTNH comprises a modification of amino acids 1062-1074 or amino acids1104-1113 of SEQ ID NO: 29.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype E NTNHcomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29, atleast 75% amino acid identity with amino acids 1062-1074 or amino acids1104-1113 of SEQ ID NO: 29, at least 80% amino acid identity with aminoacids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29, at least 85%amino acid identity with amino acids 1062-1074 or amino acids 1104-1113of SEQ ID NO: 29, at least 90% amino acid identity with amino acids1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29 or at least 95%amino acid identity with amino acids 1062-1074 or amino acids 1104-1113of SEQ ID NO: 29. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype E NTNH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1062-1074 or amino acids1104-1113 of SEQ ID NO: 29, at most 75% amino acid identity with aminoacids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29, at most 80%amino acid identity with amino acids 1062-1074 or amino acids 1104-1113of SEQ ID NO: 29, at most 85% amino acid identity with amino acids1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29, at most 90% aminoacid identity with amino acids 1062-1074 or amino acids 1104-1113 of SEQID NO: 29 or at most 95% amino acid identity with amino acids 1062-1074or amino acids 1104-1113 of SEQ ID NO: 29.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype E NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype E NTNH comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO:29 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype E NTNH comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino aciddeletions relative to amino acids 1062-1074 or amino acids 1104-1113 ofSEQ ID NO: 29. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype E NTNH comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29. In stillother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype E NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 1062-1074 oramino acids 1104-1113 of SEQ ID NO: 29. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype E NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 1062-1074 or amino acids 1104-1113 ofSEQ ID NO: 29.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype E NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 1062-1074 oramino acids 1104-1113 of SEQ ID NO: 29. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype E NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 1062-1074 or amino acids 1104-1113of SEQ ID NO: 29. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1062-1074 or aminoacids 1104-1113 of SEQ ID NO: 29 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1062-1074 or amino acids1104-1113 of SEQ ID NO: 29 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype E NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 1062-1074 oramino acids 1104-1113 of SEQ ID NO: 29. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype E NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1062-1074 or amino acids 1104-1113 ofSEQ ID NO: 29. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype E NTNH comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 1062-1074 or amino acids 1104-1113 of SEQ ID NO: 29. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype E NTNHcomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 1062-1074 oramino acids 1104-1113 of SEQ ID NO: 29.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype F NTNH comprises a β-trefoil domain derived from aClostridial botulinum serotype F NTNH of SEQ ID NO: 30. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype F NTNH comprises amino acids 1016-1160 of SEQ ID NO: 30. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype F NTNH comprises a α-fold motif of a8-trefoil domain of a Clostridial botulinum serotype F NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype F NTNHor a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype F NTNH of SEQ ID NO: 30. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype F NTNHcomprises amino acids 1016-1063, amino acids 1077-1104, or amino acids1115-1160 of SEQ ID NO: 30.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype F NTNH comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype F NTNH of SEQ ID NO: 30. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype F NTNH comprises amino acids1016-1160 of SEQ ID NO: 30. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype F NTNH comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype F NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype F NTNH or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype F NTNH of SEQ ID NO: 30. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype F NTNH comprisesamino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160of SEQ ID NO: 30.

In other aspects of this embodiment, a Clostridial botulinum serotype FNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1016-1063, amino acids 1077-1104, or amino acids1115-1160 of SEQ ID NO: 30, at least 75% amino acid identity with aminoacids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQID NO: 30, at least 80% amino acid identity with amino acids 1016-1063,amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 30, atleast 85% amino acid identity with amino acids 1016-1063, amino acids1077-1104, or amino acids 1115-1160 of SEQ ID NO: 30, at least 90% aminoacid identity with amino acids 1016-1063, amino acids 1077-1104, oramino acids 1115-1160 of SEQ ID NO: 30 or at least 95% amino acididentity with amino acids 1016-1063, amino acids 1077-1104, or aminoacids 1115-1160 of SEQ ID NO: 30. In yet other aspects of thisembodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO:30, at most 75% amino acid identity with amino acids 1016-1063, aminoacids 1077-1104, or amino acids 1115-1160 of SEQ ID NO: 30, at most 80%amino acid identity with amino acids 1016-1063, amino acids 1077-1104,or amino acids 1115-1160 of SEQ ID NO: 30, at most 85% amino acididentity with amino acids 1016-1063, amino acids 1077-1104, or aminoacids 1115-1160 of SEQ ID NO: 30, at most 90% amino acid identity withamino acids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160of SEQ ID NO: 30 or at most 95% amino acid identity with amino acids1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO:30.

In other aspects of this embodiment, a Clostridial botulinum serotype FNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1016-1063, amino acids 1077-1104,or amino acids 1115-1160 of SEQ ID NO: 30. In other aspects of thisembodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQID NO: 30. In yet other aspects of this embodiment, a Clostridialbotulinum serotype F NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 1016-1063, amino acids1077-1104, or amino acids 1115-1160 of SEQ ID NO: 30. In other aspectsof this embodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQID NO: 30. In still other aspects of this embodiment, a Clostridialbotulinum serotype F NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1016-1063, amino acids1077-1104, or amino acids 1115-1160 of SEQ ID NO: 30. In other aspectsof this embodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQID NO: 30.

In other aspects of this embodiment, a Clostridial botulinum serotype FNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1016-1063, amino acids 1077-1104,or amino acids 1115-1160 of SEQ ID NO: 30. In other aspects of thisembodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQID NO: 30. In yet other aspects of this embodiment, a Clostridialbotulinum serotype F NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 1016-1063, amino acids 1077-1104,or amino acids 1115-1160 of SEQ ID NO: 30. In other aspects of thisembodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO:30. In still other aspects of this embodiment, a Clostridial botulinumserotype F NTNH comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 1016-1063, amino acids 1077-1104, oramino acids 1115-1160 of SEQ ID NO: 30. In other aspects of thisembodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids1016-1063, amino acids 1077-1104, or amino acids 1115-1160 of SEQ ID NO:30.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype F NTNH comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype F NTNH or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype F NTNH ofSEQ ID NO: 30. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype F NTNH comprises aminoacids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype F NTNH comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype F NTNH or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype F NTNH of SEQ IDNO: 30. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeF NTNH comprises a modification of amino acids 1064-1076 or amino acids1105-1114 of SEQ ID NO: 30.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30, atleast 75% amino acid identity with amino acids 1064-1076 or amino acids1105-1114 of SEQ ID NO: 30, at least 80% amino acid identity with aminoacids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30, at least 85%amino acid identity with amino acids 1064-1076 or amino acids 1105-1114of SEQ ID NO: 30, at least 90% amino acid identity with amino acids1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30 or at least 95%amino acid identity with amino acids 1064-1076 or amino acids 1105-1114of SEQ ID NO: 30. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype F NTNH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1064-1076 or amino acids1105-1114 of SEQ ID NO: 30, at most 75% amino acid identity with aminoacids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30, at most 80%amino acid identity with amino acids 1064-1076 or amino acids 1105-1114of SEQ ID NO: 30, at most 85% amino acid identity with amino acids1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30, at most 90% aminoacid identity with amino acids 1064-1076 or amino acids 1105-1114 of SEQID NO: 30 or at most 95% amino acid identity with amino acids 1064-1076or amino acids 1105-1114 of SEQ ID NO: 30.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype F NTNH comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO:30 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype F NTNH comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino aciddeletions relative to amino acids 1064-1076 or amino acids 1105-1114 ofSEQ ID NO: 30. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype F NTNH comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30. In stillother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 1064-1076 oramino acids 1105-1114 of SEQ ID NO: 30. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype F NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 1064-1076 or amino acids 1105-1114 ofSEQ ID NO: 30.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 1064-1076 oramino acids 1105-1114 of SEQ ID NO: 30. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype F NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 1064-1076 or amino acids 1105-1114of SEQ ID NO: 30. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1064-1076 or aminoacids 1105-1114 of SEQ ID NO: 30 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1064-1076 or amino acids1105-1114 of SEQ ID NO: 30 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 1064-1076 oramino acids 1105-1114 of SEQ ID NO: 30. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype F NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1064-1076 or amino acids 1105-1114 ofSEQ ID NO: 30. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype F NTNH comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 1064-1076 or amino acids 1105-1114 of SEQ ID NO: 30. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 1064-1076 oramino acids 1105-1114 of SEQ ID NO: 30.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype F NTNH comprises a β-trefoil domain derived from aClostridial botulinum serotype F NTNH of SEQ ID NO: 31. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype F NTNH comprises amino acids 1017-1166 of SEQ ID NO: 31. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype F NTNH comprises a α-fold motif of aβ-trefoil domain of a Clostridial botulinum serotype F NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype F NTNHor a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype F NTNH of SEQ ID NO: 31. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype F NTNHcomprises amino acids 1017-1064, amino acids 1078-1106, or amino acids1117-1166 of SEQ ID NO: 31.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype F NTNH comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype F NTNH of SEQ ID NO: 31. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype F NTNH comprises amino acids1017-1166 of SEQ ID NO: 31. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype F NTNH comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype F NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype F NTNH or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype F NTNH of SEQ ID NO: 31. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype F NTNH comprisesamino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166of SEQ ID NO: 31.

In other aspects of this embodiment, a Clostridial botulinum serotype FNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1017-1064, amino acids 1078-1106, or amino acids1117-1166 of SEQ ID NO: 31, at least 75% amino acid identity with aminoacids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQID NO: 31, at least 80% amino acid identity with amino acids 1017-1064,amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 31, atleast 85% amino acid identity with amino acids 1017-1064, amino acids1078-1106, or amino acids 1117-1166 of SEQ ID NO: 31, at least 90% aminoacid identity with amino acids 1017-1064, amino acids 1078-1106, oramino acids 1117-1166 of SEQ ID NO: 31 or at least 95% amino acididentity with amino acids 1017-1064, amino acids 1078-1106, or aminoacids 1117-1166 of SEQ ID NO: 31. In yet other aspects of thisembodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO:31, at most 75% amino acid identity with amino acids 1017-1064, aminoacids 1078-1106, or amino acids 1117-1166 of SEQ ID NO: 31, at most 80%amino acid identity with amino acids 1017-1064, amino acids 1078-1106,or amino acids 1117-1166 of SEQ ID NO: 31, at most 85% amino acididentity with amino acids 1017-1064, amino acids 1078-1106, or aminoacids 1117-1166 of SEQ ID NO: 31, at most 90% amino acid identity withamino acids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166of SEQ ID NO: 31 or at most 95% amino acid identity with amino acids1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO:31.

In other aspects of this embodiment, a Clostridial botulinum serotype FNTNH comprising a 11-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1017-1064, amino acids 1078-1106,or amino acids 1117-1166 of SEQ ID NO: 31. In other aspects of thisembodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQID NO: 31. In yet other aspects of this embodiment, a Clostridialbotulinum serotype F NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 1017-1064, amino acids1078-1106, or amino acids 1117-1166 of SEQ ID NO: 31. In other aspectsof this embodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQID NO: 31. In still other aspects of this embodiment, a Clostridialbotulinum serotype F NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1017-1064, amino acids1078-1106, or amino acids 1117-1166 of SEQ ID NO: 31. In other aspectsof this embodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQID NO: 31.

In other aspects of this embodiment, a Clostridial botulinum serotype FNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1017-1064, amino acids 1078-1106,or amino acids 1117-1166 of SEQ ID NO: 31. In other aspects of thisembodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQID NO: 31. In yet other aspects of this embodiment, a Clostridialbotulinum serotype F NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 1017-1064, amino acids 1078-1106,or amino acids 1117-1166 of SEQ ID NO: 31. In other aspects of thisembodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO:31. In still other aspects of this embodiment, a Clostridial botulinumserotype F NTNH comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 1017-1064, amino acids 1078-1106, oramino acids 1117-1166 of SEQ ID NO: 31. In other aspects of thisembodiment, a Clostridial botulinum serotype F NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids1017-1064, amino acids 1078-1106, or amino acids 1117-1166 of SEQ ID NO:31.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype F NTNH comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype F NTNH or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype F NTNH ofSEQ ID NO: 31. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype F NTNH comprises aminoacids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype F NTNH comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype F NTNH or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype F NTNH of SEQ IDNO: 31. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeF NTNH comprises a modification of amino acids 1065-1077 or amino acids1107-1116 of SEQ ID NO: 31.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31, atleast 75% amino acid identity with amino acids 1065-1077 or amino acids1107-1116 of SEQ ID NO: 31, at least 80% amino acid identity with aminoacids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31, at least 85%amino acid identity with amino acids 1065-1077 or amino acids 1107-1116of SEQ ID NO: 31, at least 90% amino acid identity with amino acids1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31 or at least 95%amino acid identity with amino acids 1065-1077 or amino acids 1107-1116of SEQ ID NO: 31. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype F NTNH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1065-1077 or amino acids1107-1116 of SEQ ID NO: 31, at most 75% amino acid identity with aminoacids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31, at most 80%amino acid identity with amino acids 1065-1077 or amino acids 1107-1116of SEQ ID NO: 31, at most 85% amino acid identity with amino acids1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31, at most 90% aminoacid identity with amino acids 1065-1077 or amino acids 1107-1116 of SEQID NO: 31 or at most 95% amino acid identity with amino acids 1065-1077or amino acids 1107-1116 of SEQ ID NO: 31.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype F NTNH comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO:31 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype F NTNH comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino aciddeletions relative to amino acids 1065-1077 or amino acids 1107-1116 ofSEQ ID NO: 31. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype F NTNH comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31. In stillother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 1065-1077 oramino acids 1107-1116 of SEQ ID NO: 31. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype F NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 1065-1077 or amino acids 1107-1116 ofSEQ ID NO: 31.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 1065-1077 oramino acids 1107-1116 of SEQ ID NO: 31. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype F NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 1065-1077 or amino acids 1107-1116of SEQ ID NO: 31. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1065-1077 or aminoacids 1107-1116 of SEQ ID NO: 31 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1065-1077 or amino acids1107-1116 of SEQ ID NO: 31 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 1065-1077 oramino acids 1107-1116 of SEQ ID NO: 31. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype F NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1065-1077 or amino acids 1107-1116 ofSEQ ID NO: 31. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype F NTNH comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 1065-1077 or amino acids 1107-1116 of SEQ ID NO: 31. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype F NTNHcomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 1065-1077 oramino acids 1107-1116 of SEQ ID NO: 31.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype G NTNH comprises a β-trefoil domain derived from aClostridial botulinum serotype G NTNH of SEQ ID NO: 32. In anotherembodiment, a β-trefoil domain derived from a Clostridial botulinumserotype G NTNH comprises amino acids 1050-1199 of SEQ ID NO: 32. Inanother aspect of this embodiment, a β-trefoil domain derived from aClostridial botulinum serotype G NTNH comprises a α-fold motif of a8-trefoil domain of a Clostridial botulinum serotype G NTNH, a β-foldmotif of a β-trefoil domain of a Clostridial botulinum serotype G NTNHor a γ-fold motif of a β-trefoil domain of a Clostridial botulinumserotype G NTNH of SEQ ID NO: 32. In another aspect of this embodiment,a β-trefoil domain derived from a Clostridial botulinum serotype G NTNHcomprises amino acids 1050-1097, amino acids 1111-1139, or amino acids1150-1199 of SEQ ID NO: 32.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype G NTNH comprises aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype G NTNH of SEQ ID NO: 32. In anotherembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype G NTNH comprises amino acids1050-1199 of SEQ ID NO: 32. In another aspect of this embodiment, aβ-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype G NTNH comprises a modified α-fold motifof a β-trefoil domain of a Clostridial botulinum serotype G NTNH, amodified β-fold motif of a β-trefoil domain of a Clostridial botulinumserotype G NTNH or a modified γ-fold motif of a β-trefoil domain of aClostridial botulinum serotype G NTNH of SEQ ID NO: 32. In anotheraspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a Clostridial botulinum serotype G NTNH comprisesamino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199of SEQ ID NO: 32.

In other aspects of this embodiment, a Clostridial botulinum serotype GNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1050-1097, amino acids 1111-1139, or amino acids1150-1199 of SEQ ID NO: 32, at least 75% amino acid identity with aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQID NO: 32, at least 80% amino acid identity with amino acids 1050-1097,amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 32, atleast 85% amino acid identity with amino acids 1050-1097, amino acids1111-1139, or amino acids 1150-1199 of SEQ ID NO: 32, at least 90% aminoacid identity with amino acids 1050-1097, amino acids 1111-1139, oramino acids 1150-1199 of SEQ ID NO: 32 or at least 95% amino acididentity with amino acids 1050-1097, amino acids 1111-1139, or aminoacids 1150-1199 of SEQ ID NO: 32. In yet other aspects of thisembodiment, a Clostridial botulinum serotype G NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO:32, at most 75% amino acid identity with amino acids 1050-1097, aminoacids 1111-1139, or amino acids 1150-1199 of SEQ ID NO: 32, at most 80%amino acid identity with amino acids 1050-1097, amino acids 1111-1139,or amino acids 1150-1199 of SEQ ID NO: 32, at most 85% amino acididentity with amino acids 1050-1097, amino acids 1111-1139, or aminoacids 1150-1199 of SEQ ID NO: 32, at most 90% amino acid identity withamino acids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199of SEQ ID NO: 32 or at most 95% amino acid identity with amino acids1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO:32.

In other aspects of this embodiment, a Clostridial botulinum serotype GNTNH comprising a 11-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidsubstitutions relative to amino acids 1050-1097, amino acids 1111-1139,or amino acids 1150-1199 of SEQ ID NO: 32. In other aspects of thisembodiment, a Clostridial botulinum serotype G NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid substitutions relative to aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQID NO: 32. In yet other aspects of this embodiment, a Clostridialbotulinum serotype G NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 1050-1097, amino acids1111-1139, or amino acids 1150-1199 of SEQ ID NO: 32. In other aspectsof this embodiment, a Clostridial botulinum serotype G NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid deletions relative to aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQID NO: 32. In still other aspects of this embodiment, a Clostridialbotulinum serotype G NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid additions relative to amino acids 1050-1097, amino acids1111-1139, or amino acids 1150-1199 of SEQ ID NO: 32. In other aspectsof this embodiment, a Clostridial botulinum serotype G NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 non-contiguous amino acid additions relative to aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQID NO: 32.

In other aspects of this embodiment, a Clostridial botulinum serotype GNTNH comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acidsubstitutions relative to amino acids 1050-1097, amino acids 1111-1139,or amino acids 1150-1199 of SEQ ID NO: 32. In other aspects of thisembodiment, a Clostridial botulinum serotype G NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid substitutions relative to aminoacids 1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQID NO: 32. In yet other aspects of this embodiment, a Clostridialbotulinum serotype G NTNH comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid deletions relative to amino acids 1050-1097, amino acids 1111-1139,or amino acids 1150-1199 of SEQ ID NO: 32. In other aspects of thisembodiment, a Clostridial botulinum serotype G NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid deletions relative to amino acids1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO:32. In still other aspects of this embodiment, a Clostridial botulinumserotype G NTNH comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 1050-1097, amino acids 1111-1139, oramino acids 1150-1199 of SEQ ID NO: 32. In other aspects of thisembodiment, a Clostridial botulinum serotype G NTNH comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at least one, two, three, four, five, six, seven, eight,nine, 10 or 20 contiguous amino acid additions relative to amino acids1050-1097, amino acids 1111-1139, or amino acids 1150-1199 of SEQ ID NO:32.

In another embodiment, a β-trefoil domain derived from a Clostridialbotulinum serotype G NTNH comprises a β4/β5 hairpin turn of a β-trefoildomain of a Clostridial botulinum serotype G NTNH or a β8/β9 hairpinturn of a β-trefoil domain of a Clostridial botulinum serotype G NTNH ofSEQ ID NO: 32. In another aspect of this embodiment, a β-trefoil domainderived from a Clostridial botulinum serotype G NTNH comprises aminoacids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype G NTNH comprises amodified β4/β5 hairpin turn of a β-trefoil domain of a Clostridialbotulinum serotype G NTNH or a modified β8/β9 hairpin turn of aβ-trefoil domain of a Clostridial botulinum serotype G NTNH of SEQ IDNO: 32. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a Clostridial botulinum serotypeG NTNH comprises a modification of amino acids 1098-1110 or amino acids1140-1149 of SEQ ID NO: 32.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype G NTNHcomprises a polypeptide having, e.g., at least 70% amino acid identitywith amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32, atleast 75% amino acid identity with amino acids 1098-1110 or amino acids1140-1149 of SEQ ID NO: 32, at least 80% amino acid identity with aminoacids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32, at least 85%amino acid identity with amino acids 1098-1110 or amino acids 1140-1149of SEQ ID NO: 32, at least 90% amino acid identity with amino acids1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32 or at least 95%amino acid identity with amino acids 1098-1110 or amino acids 1140-1149of SEQ ID NO: 32. In yet other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype G NTNH comprises a polypeptide having, e.g., at most70% amino acid identity with amino acids 1098-1110 or amino acids1140-1149 of SEQ ID NO: 32, at most 75% amino acid identity with aminoacids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32, at most 80%amino acid identity with amino acids 1098-1110 or amino acids 1140-1149of SEQ ID NO: 32, at most 85% amino acid identity with amino acids1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32, at most 90% aminoacid identity with amino acids 1098-1110 or amino acids 1140-1149 of SEQID NO: 32 or at most 95% amino acid identity with amino acids 1098-1110or amino acids 1140-1149 of SEQ ID NO: 32.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype G NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32. In other aspects ofthis embodiment, a β-trefoil domain with enhanced binding activityderived from a Clostridial botulinum serotype G NTNH comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32. In other aspects ofthis embodiment, a non-contiguous amino acid substitution of any aminoacid from amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO:32 can be replaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32 canbe replaced with phenylalanine. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from aClostridial botulinum serotype G NTNH comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1140-1149 ofSEQ ID NO: 32. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a Clostridial botulinumserotype G NTNH comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32. In stillother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype G NTNHcomprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid additions relative to amino acids 1098-1110 oramino acids 1140-1149 of SEQ ID NO: 32. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype G NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 1098-1110 or amino acids 1140-1149 ofSEQ ID NO: 32.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype G NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid substitutions relative to amino acids 1098-1110 oramino acids 1140-1149 of SEQ ID NO: 32. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype G NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidsubstitutions relative to amino acids 1098-1110 or amino acids 1140-1149of SEQ ID NO: 32. In other aspects of this embodiment, contiguous aminoacid substitutions of amino acids from amino acids 1098-1110 or aminoacids 1140-1149 of SEQ ID NO: 32 can be replaced with glycine. In otheraspects of this embodiment, contiguous amino acid substitutions ofhydrophobic amino acids from amino acids 1098-1110 or amino acids1140-1149 of SEQ ID NO: 32 can be replaced with phenylalanine. In yetother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype G NTNHcomprises a polypeptide having, e.g., at most one, two, three or fourcontiguous amino acid deletions relative to amino acids 1098-1110 oramino acids 1140-1149 of SEQ ID NO: 32. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a Clostridial botulinum serotype G NTNH comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 1098-1110 or amino acids 1140-1149 ofSEQ ID NO: 32. In still other aspects of this embodiment, a β-trefoildomain with enhanced binding activity derived from a Clostridialbotulinum serotype G NTNH comprises a polypeptide having, e.g., at mostone, two, three or four contiguous amino acid additions relative toamino acids 1098-1110 or amino acids 1140-1149 of SEQ ID NO: 32. Inother aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a Clostridial botulinum serotype G NTNHcomprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 1098-1110 oramino acids 1140-1149 of SEQ ID NO: 32.

Another example of an enhanced targeting domain that increases bindingactivity for an endogenous Clostridial toxin receptor present on anaturally-occurring Clostridial toxin target cell, includes, withoutlimitation, ligands that bind the same receptors as naturally-occurringClostridial toxins. Recent studies are revealing components of theendogenous receptors used by a Clostridial toxin during the intoxicationprocess. As a non-limiting example, fibroblast growth factor 3 receptor(FGFR3) serves as a BoNT/A receptor, see, e.g., Ester Fernandez-Salas etal., Botulinum Toxin Screening Assays, PCT Patent Application No.2005/006421 (Sep. 9, 2005). As another non-limiting example,synaptotagmin I serves as a BoNT/B receptor and as a BoNT/G receptor,see, e.g., Min Dong et al., Synaptotagmins I and II mediate entry ofbotulinum neurotoxin B into cells, 162(7) J. Cell Biol. 1293-1303(2003); and Andreas Rummel et al., Synaptotagmins I and II act as nervecell receptors for botulinum neurotoxin G, 279(29) J. Biol. Chem.30865-30870 (2004). As yet another non-limiting example, synaptotagminII serves as a BoNT/B receptor and as a BoNT/G receptor, see, e.g., MinDong et al., supra, (2003); and Andreas Rummel et al., supra, (2004).The selection of a ligand domain as an enhanced binding region willdepend on the Clostridial toxin being modified. As non-limitingexamples, ligands that selectively bind to a FGFR3 could be used as anenhanced targeting domain for a modified BoNT/A, whereas ligands thatselectively bind a Synaptotagmin I or Synaptotagmin II could be used asan enhanced targeting domain for a modified BoNT/B or a modified BoNT/G.

In addition to its enhanced targeting activity, replacement of anaturally-occurring targeting domain with an FGF ligand has an addedadvantage of reducing the likelihood of the modified toxin fromeliciting an immunogenic response. Regions found in the H_(CC) targetingdomain are bound by neutralizing anti-BoNT/A antibodies, see, e.g., M.Zouhair Atassi et al., Mapping of the Antibody-binding Regions onBotulinum Neurotoxin H-chain Domain 855-1296 with Anti-toxin Antibodiesfrom Three Host Species, 15 J. PROT. CHEM. 691-700, (1996); M. ZouhairAtassi & Behzod Z. Dolimbek, Mapping of the Antibody-binding Profile onBotulinum Neurotoxin A H _(N)-domain (residues 449-859) with Anti-toxinAntibodies from Four Host Species. Antigenic Profile of the EntireH-chain of Botulinum Neurotoxin A, 23(1) PROTEIN J. 39-52, (2004).Therefore, elimination of this targeting domain will reduce thelikelihood of an immunogenic response because 1) the Clostridial toxinH_(CC) targeting domain is absent; 2) a human FGF targeting domain willmost likely not elicit an immunogenic response in a patient because itis a human polypeptide.

Fibroblast growth factors (FGF) participate in many developmental,differentiation and growth and repair processes of cells through complexcombinatorial signaling pathways. Presently, at least 23 ligands(FGF1-23) are known to signal through a family of five transmembranetyrosine kinase FGF receptors (FGFR1-5). Affinity of FGFRs for theirligands is highly diverse with different affinities for each familymember of growth factors, see, e.g., C. J. Powers et al., Fibroblastgrowth factors, their receptors and signaling, 7(3) Endocr. Relat.Cancer. 165-197 (2000). This diversity is achieved in part by thegeneration of alternatively spliced variants encoding distinct receptorisoforms, see, e.g., Bernhard Reuss & Oliver von Bohlen and Halbach,Fibroblast growth factors and their receptors in the central nervoussystem, 313(2) Cell Tissue Res. 139-157 (2003). The protein region thatappears to have the highest influence on ligand binding selectivity is aportion of the 1011 domain, for which isoforms encoded by threedifferent splice variants have been identified. These three isoforms,designated IgIIIa, IgIIIb and IgIIIc, have relative binding affinitiesfor different FGFR family members. Alternative splicing in the FGFRligand binding domain, designated a and b, generates additional receptorisoforms with novel ligand affinities. Isoforms for IgIIIa, IgIIIb andIgIIIc have been identified for both FGFR1 and FGFR2. Thus far, theIgIIIa isoform of FGFR3 and the IgIIIa and IgIIIb isoforms of FGFR4 andFGFR5 have not been reported.

Currently, several FGFs have been shown to selectively bind FGFR3, suchas, e.g., FGF-1, FGF-2, FGF-4, FGF-8, FGF-9, FGF-17 and FGF-18, see,e.g., Hecht et al., 1995; Ornitz et al., 1996; and Xu et al., 2000.Additional studies have revealed that FGF-1 and FGF9 preferentially bindFGFR3IIIb, whereas FGF-1 FGF-2, FGF-4, FGF-8, and FGF9 preferentiallybind FGFR3IIIc. Studies have shown that each of these ligands is presentin various vertebrate species with a high degree of sequence identitywhich suggests functional equivalence or similarity. As a non-limitingexample, FGF-8 is found in fish, birds and mammals and each of theseFGF-8 ligands have over 80% amino acid sequence identity. As anothernon-limiting example, FGF-18 is found in fish, birds and mammals andeach of these FGF-18 ligands have over 70% amino acid sequence identity.Crystallographic studies have revealed that all FGFs are structurallyorganized into a β-trefoil domain. The amino acid sequences comprisingthe β-trefoil domains found in various FGF ligands that bind FGFR3 areshown in Table 7.

TABLE 7 β-trefoil Domains of FGFs Amino Acid Sequence Region ofCarbohydrate Binding Moieties β4/β5 β8/β9 Ligand SEQ ID NO: α-foldβ-hairpin turn β-fold β-hairpin turn γ-fold FGF-1 33 26-64 65-67 68-105106-108 109-155 FGF-2 34 29-67 68-70 71-111 112-114 115-155 FGF-4 35 83-121 122-124 125-162  163-165 166-206 FGF-8 36 43-80 81-83 84-123124-126 127-172 FGF-9 37  63-100 101-103 104-144  145-147 148-196 FGF-1738 55-91 92-94 95-134 135-137 138-183 FGF-18 39 54-91 92-94 95-134135-137 138-183

As used herein, the term “Fibroblast Growth Factor” is synonymous with“FGF” and means a polypeptide with selective binding activity, such as,e.g., a binding affinity or a binding specificity, for a receptor,including endogenous Clostridial toxin receptors such as, e.g., areceptor comprising FGFR3. It is envisioned that both naturallyoccurring FGFs as well as FGFs with enhanced binding activity can beused to practice aspects of the present invention. As used herein, theterm “FGF with enhanced binding activity” means a FGF with enhancedbinding activity for an endogenous Clostridial toxin receptor, such as,e.g., a binding affinity or a binding specificity, to a statisticallysignificantly degree relative to an unmodified naturally occurringClostridial toxin binding domain from a Clostridial toxin. Bydefinition, a FGF with enhanced binding activity has at least one aminoacid change from the corresponding region of the disclosed referencesequences (see Table 7) and can be described in percent identity to thecorresponding region of that reference sequence.

Any of a variety of sequence alignment methods can be used to determinepercent identity of a modified FGF relative to a naturally-occurringFGF, including, without limitation, global methods, local methods andhybrid methods, such as, e.g., segment approach methods. Protocols todetermine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

Approaches well known to one skilled in the art on how to modify a FGFin order to increase its binding activity for an endogenous Clostridialtoxin receptor present on a naturally-occurring Clostridial toxin targetcell. As described above, one approach involves identifying amino acidsusing computational protein design algorithms; changingspecifically-identified amino acids using, without limitation,site-directed mutagenesis, oligonucleotide-directed mutagenesis andsite-specific mutagenesis; and testing the binding activity of modifiedClostridial toxins comprising a modified FGF with enhanced bindingactivity using, e.g., heterogeneous assays, homogeneous assays andnon-separating homogeneous assays. It is further envisioned that thebinding activity of a modified Clostridial toxin with enhanced bindingactivity disclosed in the present specification can be determined byaffinity chromatography using immobilized receptors and interfacialoptical assays. In another approach described above, a binding activityof a modified FGF for a naturally-occurring Clostridial toxin receptorpresent on a naturally-occurring Clostridial toxin target cell can beachieved using directed-evolution methods.

A FGF includes, without limitation, naturally occurring FGF variants,such as, e.g., FGF isoforms and FGF subtypes; non-naturally occurringFGF variants, such as, e.g., conservative FGF variants, non-conservativeFGF variants, FGF chimerics, active FGF fragments thereof, or anycombination thereof.

As used herein, the term “FGF variant,” whether naturally-occurring ornon-naturally-occurring, means a FGF that has at least one amino acidchange from the corresponding region of the disclosed referencesequences (see Tables 7) and can be described in percent identity to thecorresponding region of that reference sequence. Unless expresslyindicated, all FGF variants disclosed in the present specification arecapable of executing the cell binding step of the intoxication process.

It is recognized by those of skill in the art that within eachClostridial bacterium there can be naturally occurring FGF variants thatdiffer somewhat in their amino acid sequence, and also in the nucleicacids encoding these proteins. For example, there are presently at leastfour FGF-8 variants, FGF-8A, FGF-8A, FGF-8B, FGF-8E and FGF-8F, withspecific FGF-8 variants showing various degrees of amino acid divergencewhen compared to another FGF-8 variant. As another example, there arepresently at least two FGF-2 variants, FGF-2-1 and FGF-2-1, with theFGF-2-1 variant showing an amino acid divergence when compared to theFGF-2-2 variant. As used herein, the term “naturally occurring FGFvariant” means any FGF produced by a naturally-occurring process,including, without limitation, FGF isoforms produced fromalternatively-spliced transcripts, FGF isoforms produced by spontaneousmutation and FGF subtypes. A naturally occurring FGF variant canfunction in substantially the same manner as the reference FGF on whichthe naturally occurring FGF variant is based, and can be substituted forthe reference FGF in any aspect of the present invention. A naturallyoccurring FGF variant may substitute one or more amino acids, two ormore amino acids, three or more amino acids, four or more amino acids,five or more amino acids, ten or more amino acids, 20 or more aminoacids, 30 or more amino acids, 40 or more amino acids, 50 or more aminoacids or 100 or more amino acids from the reference FGF on which thenaturally occurring FGF variant is based. A naturally occurring FGFvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference FGF on which thenaturally occurring FGF variant is based, that possess at least 50%amino acid identity, 65% amino acid identity, 75% amino acid identity,85% amino acid identity or 95% amino acid identity to the reference FGFon which the naturally occurring FGF variant is based.

A non-limiting examples of a naturally occurring FGF variant is a FGFisoform such as, e.g., a FGF-1 isoform, a FGF-2 isoform, a FGF-4isoform, a FGF-8 isoform, a FGF-9 isoform, a FGF-17 isoform and a FGF-18isoform. A FGF isoform can function in substantially the same manner asthe reference FGF on which the FGF isoform is based, and can besubstituted for the reference FGF in any aspect of the presentinvention.

Another non-limiting examples of a naturally occurring FGF variant is aFGF subtype such as, e.g., a FGF-1 subtype, a FGF-2 subtype, a FGF-4subtype, a FGF-8 subtype, a FGF-9 subtype, a FGF-17 subtype and a FGF-18subtype. A FGF subtype can function in substantially the same manner asthe reference FGF on which the FGF subtype is based, and can besubstituted for the reference FGF in any aspect of the presentinvention.

As used herein, the term “non-naturally occurring FGF variant” means anyFGF produced with the aid of human manipulation, including, withoutlimitation, FGFs produced by genetic engineering using randommutagenesis or rational design and FGFs produced by chemical synthesis.Non-limiting examples of non-naturally occurring FGF variants include,e.g., conservative FGF variants, non-conservative FGF variants, FGFchimeric variants and active FGF fragments.

As used herein, the term “conservative FGF variant” means a FGF that hasat least one amino acid substituted by another amino acid or an aminoacid analog that has at least one property similar to that of theoriginal amino acid from the reference FGF sequence (see Table 7).Examples of properties include, without limitation, similar size,topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservative FGFvariant can function in substantially the same manner as the referenceFGF on which the conservative FGF variant is based, and can besubstituted for the reference FGF in any aspect of the presentinvention. A conservative FGF variant may substitute one or more aminoacids, two or more amino acids, three or more amino acids, four or moreamino acids, five or more amino acids, ten or more amino acids, 20 ormore amino acids, 30 or more amino acids, 40 or more amino acids or 50or more amino acids from the reference FGF on which the conservative FGFvariant is based. A conservative FGF variant can also substitute atleast 10 contiguous amino acids, at least 15 contiguous amino acids, atleast 20 contiguous amino acids, or at least 25 contiguous amino acidsfrom the reference FGF on which the conservative FGF variant is based,that possess at least 50% amino acid identity, 65% amino acid identity,75% amino acid identity, 85% amino acid identity or 95% amino acididentity to the reference FGF on which the conservative FGF variant isbased. Non-limiting examples of a conservative FGF variant include,e.g., a conservative FGF-1 variant, a conservative FGF-2 variant, aconservative FGF-4 variant, a conservative FGF-8 variant, a conservativeFGF-9 variant, a conservative FGF-17 variant and a conservative FGF-18variant.

As used herein, the term “non-conservative FGF variant” means a FGF inwhich 1) at least one amino acid is deleted from the reference FGF onwhich the non-conservative FGF variant is based; 2) at least one aminoacid added to the reference FGF on which the non-conservative FGF isbased; or 3) at least one amino acid is substituted by another aminoacid or an amino acid analog that does not share any property similar tothat of the original amino acid from the reference FGF sequence (seeTable 7). A non-conservative FGF variant can function in substantiallythe same manner as the reference FGF on which the non-conservative FGFvariant is based, and can be substituted for the reference FGF in anyaspect of the present invention. A non-conservative FGF variant candelete one or more amino acids, two or more amino acids, three or moreamino acids, four or more amino acids, five or more amino acids, and tenor more amino acids from the reference FGF on which the non-conservativeFGF variant is based. A non-conservative FGF variant can add one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, and ten or more amino acidsto the reference FGF on which the non-conservative FGF variant is based.A non-conservative FGF variant may substitute one or more amino acids,two or more amino acids, three or more amino acids, four or more aminoacids, five or more amino acids, ten or more amino acids, 20 or moreamino acids, 30 or more amino acids, 40 or more amino acids or 50 ormore amino acids from the reference FGF on which the non-conservativeFGF variant is based. A non-conservative FGF variant can also substituteat least 10 contiguous amino acids, at least 15 contiguous amino acids,at least 20 contiguous amino acids, or at least 25 contiguous aminoacids from the reference FGF on which the non-conservative FGF variantis based, that possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference FGF on which the non-conservative FGFvariant is based. Non-limiting examples of a non-conservative FGFvariant include, e.g., a non-conservative FGF-1 variant, anon-conservative FGF-2 variant, a non-conservative FGF-4 variant, anon-conservative FGF-8 variant, a non-conservative FGF-9 variant, anon-conservative FGF-17 variant and a non-conservative FGF-18 variant.

As used herein, the term “FGF chimeric” means a polypeptide comprisingat least a portion of a FGF and at least a portion of at least one otherpolypeptide to form an enhanced targeting domain with at least oneproperty different from the reference FGF (see Tables 7), with theproviso that this FGF chimeric can specifically bind to an endogenousClostridial toxin receptor present in a Clostridial toxin target cell,and thus participate in executing the overall cellular mechanism wherebya Clostridial toxin proteolytically cleaves a substrate.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises an enhanced targeting domain comprisinga β-trefoil domain derived from a FGF. In an aspect of this embodiment,a β-trefoil domain derived from a FGF comprises, e.g., a β-trefoildomain derived from a FGF-1, a β-trefoil domain derived from a FGF-2, aβ-trefoil domain derived from FGF-4, a β-trefoil domain derived from aFGF-8, a β-trefoil domain derived from a FGF-9, a β-trefoil domainderived from a FGF-17 or a β-trefoil domain derived from a FGF-18. Inanother aspect of this embodiment, a β-trefoil domain derived from a FGFcomprises a α-fold motif of a β-trefoil domain of a FGF, a β-fold motifof a β-trefoil domain of a FGF or a γ-fold motif of a β-trefoil domainof a FGF.

In an embodiment, a modified Clostridial toxin disclosed in the presentspecification comprises an enhanced targeting domain comprising aβ-trefoil domain with enhanced binding activity derived from a FGF. Inan aspect of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a FGF comprises, e.g., a β-trefoil domain withenhanced binding activity derived from a FGF-1, a β-trefoil domain withenhanced binding activity derived from a FGF-2, a β-trefoil domain withenhanced binding activity derived from a FGF-4, a β-trefoil domain withenhanced binding activity derived from a FGF-8, a β-trefoil domain withenhanced binding activity derived from a FGF-9, a β-trefoil domain withenhanced binding activity derived from a FGF-17 or a β-trefoil domainwith enhanced binding activity derived from a FGF-18. In another aspectof this embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF comprises a modified α-fold motif of a β-trefoildomain of a FGF, a modified β-fold motif of a β-trefoil domain of a FGFor a modified γ-fold motif of a β-trefoil domain of a FGF.

In another embodiment, a β-trefoil domain derived from a FGF-1 comprisesa β-trefoil domain derived from a FGF-1 of SEQ ID NO: 33. In anotherembodiment, a β-trefoil domain derived from a FGF-1 comprises aminoacids 26-155 of SEQ ID NO: 33. In another aspect of this embodiment, aβ-trefoil domain derived from a FGF-1 comprises a α-fold motif of aβ-trefoil domain of a FGF-1, a β-fold motif of a β-trefoil domain of aFGF-1 or a γ-fold motif of a β-trefoil domain of a FGF-1 of SEQ ID NO:33. In another aspect of this embodiment, a β-trefoil domain derivedfrom a FGF-1 comprises amino acids 26-64, amino acids 68-105, or aminoacids 109-155 of SEQ ID NO: 33.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-1 comprises a β-trefoil domain with enhanced bindingactivity derived from a FGF-1 of SEQ ID NO: 33. In another embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-1comprises amino acids 26-155 of SEQ ID NO: 33. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-1 comprises a modified α-fold motif of a β-trefoil domain ofa FGF-1, a modified β-fold motif of a β-trefoil domain of a FGF-1 or amodified γ-fold motif of a β-trefoil domain of a FGF-1 of SEQ ID NO: 33.In another aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-1 comprises amino acids 26-64, aminoacids 68-105, or amino acids 109-155 of SEQ ID NO: 33.

In other aspects of this embodiment, a FGF-1 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 26-64, aminoacids 68-105, or amino acids 109-155 of SEQ ID NO: 33, at least 75%amino acid identity with amino acids 26-64, amino acids 68-105, or aminoacids 109-155 of SEQ ID NO: 33, at least 80% amino acid identity withamino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ IDNO: 33, at least 85% amino acid identity with amino acids 26-64, aminoacids 68-105, or amino acids 109-155 of SEQ ID NO: 33, at least 90%amino acid identity with amino acids 26-64, amino acids 68-105, or aminoacids 109-155 of SEQ ID NO: 33 or at least 95% amino acid identity withamino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ IDNO: 33. In yet other aspects of this embodiment, a FGF-1 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 26-64,amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 33, at most 75%amino acid identity with amino acids 26-64, amino acids 68-105, or aminoacids 109-155 of SEQ ID NO: 33, at most 80% amino acid identity withamino acids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ IDNO: 33, at most 85% amino acid identity with amino acids 26-64, aminoacids 68-105, or amino acids 109-155 of SEQ ID NO: 33, at most 90% aminoacid identity with amino acids 26-64, amino acids 68-105, or amino acids109-155 of SEQ ID NO: 33 or at most 95% amino acid identity with aminoacids 26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO:33.

In other aspects of this embodiment, a FGF-1 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid substitutions relative to amino acids26-64, amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 33. Inother aspects of this embodiment, a FGF-1 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 26-64,amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 33. In yetother aspects of this embodiment, a FGF-1 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 26-64, aminoacids 68-105, or amino acids 109-155 of SEQ ID NO: 33. In other aspectsof this embodiment, a FGF-1 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 26-64, amino acids 68-105,or amino acids 109-155 of SEQ ID NO: 33. In still other aspects of thisembodiment, a FGF-1 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 26-64, amino acids 68-105, or aminoacids 109-155 of SEQ ID NO: 33. In other aspects of this embodiment, aFGF-1 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 26-64, amino acids 68-105, or aminoacids 109-155 of SEQ ID NO: 33.

In other aspects of this embodiment, a FGF-1 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid substitutions relative to amino acids 26-64,amino acids 68-105, or amino acids 109-155 of SEQ ID NO: 33. In otheraspects of this embodiment, a FGF-1 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 26-64, aminoacids 68-105, or amino acids 109-155 of SEQ ID NO: 33. In yet otheraspects of this embodiment, a FGF-1 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 26-64, aminoacids 68-105, or amino acids 109-155 of SEQ ID NO: 33. In other aspectsof this embodiment, a FGF-1 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 26-64, amino acids 68-105,or amino acids 109-155 of SEQ ID NO: 33. In still other aspects of thisembodiment, a FGF-1 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 26-64, amino acids 68-105, or aminoacids 109-155 of SEQ ID NO: 33. In other aspects of this embodiment, aFGF-1 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 26-64, amino acids 68-105, or amino acids109-155 of SEQ ID NO: 33.

In another embodiment, a β-trefoil domain derived from a FGF-1 comprisesa β4/β5 hairpin turn of a β-trefoil domain of a FGF-1 or a β8/β9 hairpinturn of a β-trefoil domain of a FGF-1 of SEQ ID NO: 33. In anotheraspect of this embodiment, a β-trefoil domain derived from a FGF-1comprises amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 33.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-1 comprises a modified β4/β5 hairpin turn of aβ-trefoil domain of a FGF-1 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a FGF-1 of SEQ ID NO: 33. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-1 comprises a modification of amino acids 65-67 or aminoacids 106-108 of SEQ ID NO: 33.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-1 comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 65-67 or aminoacids 106-108 of SEQ ID NO: 33, at least 75% amino acid identity withamino acids 65-67 or amino acids 106-108 of SEQ ID NO: 33, at least 80%amino acid identity with amino acids 65-67 or amino acids 106-108 of SEQID NO: 33, at least 85% amino acid identity with amino acids 65-67 oramino acids 106-108 of SEQ ID NO: 33, at least 90% amino acid identitywith amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 33 or atleast 95% amino acid identity with amino acids 65-67 or amino acids106-108 of SEQ ID NO: 33. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-1comprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 33, at most75% amino acid identity with amino acids 65-67 or amino acids 106-108 ofSEQ ID NO: 33, at most 80% amino acid identity with amino acids 65-67 oramino acids 106-108 of SEQ ID NO: 33, at most 85% amino acid identitywith amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 33, at most90% amino acid identity with amino acids 65-67 or amino acids 106-108 ofSEQ ID NO: 33 or at most 95% amino acid identity with amino acids 65-67or amino acids 106-108 of SEQ ID NO: 33.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-1 comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 65-67 or amino acids 106-108 ofSEQ ID NO: 33. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-1 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 65-67 oramino acids 106-108 of SEQ ID NO: 33. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 33 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 33 can bereplaced with phenylalanine. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-1comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 65-67 oramino acids 106-108 of SEQ ID NO: 33. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-1 comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 65-67 or amino acids 106-108 of SEQ ID NO: 33. In still otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a FGF-1 comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid additionsrelative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 33.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-1 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 65-67 or amino acids 106-108 of SEQ IDNO: 33.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-1 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acidsubstitutions relative to amino acids 65-67 or amino acids 106-108 ofSEQ ID NO: 33. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-1 comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid substitutions relative to amino acids 65-67 or amino acids106-108 of SEQ ID NO: 33. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids65-67 or amino acids 106-108 of SEQ ID NO: 33 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 65-67 or aminoacids 106-108 of SEQ ID NO: 33 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-1 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 33.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-1 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acid deletionsrelative to amino acids 65-67 or amino acids 106-108 of SEQ ID NO: 33.In still other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-1 comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 65-67 or amino acids 106-108 of SEQ IDNO: 33. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-1 comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 65-67 or amino acids 106-108 of SEQ IDNO: 33.

In another embodiment, a β-trefoil domain derived from a FGF-2 comprisesa β-trefoil domain derived from a FGF-2 of SEQ ID NO: 34. In anotherembodiment, a β-trefoil domain derived from a FGF-2 comprises aminoacids 29-155 of SEQ ID NO: 34. In another aspect of this embodiment, aβ-trefoil domain derived from a FGF-2 comprises a α-fold motif of aβ-trefoil domain of a FGF-2, a β-fold motif of a β-trefoil domain of aFGF-2 or a γ-fold motif of a β-trefoil domain of a FGF-2 of SEQ ID NO:34. In another aspect of this embodiment, a β-trefoil domain derivedfrom a FGF-2 comprises amino acids 29-67, amino acids 71-111, or aminoacids 115-155 of SEQ ID NO: 34.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-2 comprises a β-trefoil domain with enhanced bindingactivity derived from a FGF-2 of SEQ ID NO: 34. In another embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-2comprises amino acids 29-155 of SEQ ID NO: 34. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-2 comprises a modified α-fold motif of a β-trefoil domain ofa FGF-2, a modified β-fold motif of a β-trefoil domain of a FGF-2 or amodified γ-fold motif of a β-trefoil domain of a FGF-2 of SEQ ID NO: 34.In another aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-2 comprises amino acids 29-67, aminoacids 71-111, or amino acids 115-155 of SEQ ID NO: 34.

In other aspects of this embodiment, a FGF-2 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 29-67, aminoacids 71-111, or amino acids 115-155 of SEQ ID NO: 34, at least 75%amino acid identity with amino acids 29-67, amino acids 71-111, or aminoacids 115-155 of SEQ ID NO: 34, at least 80% amino acid identity withamino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ IDNO: 34, at least 85% amino acid identity with amino acids 29-67, aminoacids 71-111, or amino acids 115-155 of SEQ ID NO: 34, at least 90%amino acid identity with amino acids 29-67, amino acids 71-111, or aminoacids 115-155 of SEQ ID NO: 34 or at least 95% amino acid identity withamino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ IDNO: 34. In yet other aspects of this embodiment, a FGF-2 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 29-67,amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 34, at most 75%amino acid identity with amino acids 29-67, amino acids 71-111, or aminoacids 115-155 of SEQ ID NO: 34, at most 80% amino acid identity withamino acids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ IDNO: 34, at most 85% amino acid identity with amino acids 29-67, aminoacids 71-111, or amino acids 115-155 of SEQ ID NO: 34, at most 90% aminoacid identity with amino acids 29-67, amino acids 71-111, or amino acids115-155 of SEQ ID NO: 34 or at most 95% amino acid identity with aminoacids 29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO:34.

In other aspects of this embodiment, a FGF-2 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid substitutions relative to amino acids29-67, amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 34. Inother aspects of this embodiment, a FGF-2 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 29-67,amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 34. In yetother aspects of this embodiment, a FGF-2 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 29-67, aminoacids 71-111, or amino acids 115-155 of SEQ ID NO: 34. In other aspectsof this embodiment, a FGF-2 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 29-67, amino acids 71-111,or amino acids 115-155 of SEQ ID NO: 34. In still other aspects of thisembodiment, a FGF-2 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 29-67, amino acids 71-111, or aminoacids 115-155 of SEQ ID NO: 34. In other aspects of this embodiment, aFGF-2 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 29-67, amino acids 71-111, or aminoacids 115-155 of SEQ ID NO: 34.

In other aspects of this embodiment, a FGF-2 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid substitutions relative to amino acids 29-67,amino acids 71-111, or amino acids 115-155 of SEQ ID NO: 34. In otheraspects of this embodiment, a FGF-2 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 29-67, aminoacids 71-111, or amino acids 115-155 of SEQ ID NO: 34. In yet otheraspects of this embodiment, a FGF-2 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 29-67, aminoacids 71-111, or amino acids 115-155 of SEQ ID NO: 34. In other aspectsof this embodiment, a FGF-2 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 29-67, amino acids 71-111,or amino acids 115-155 of SEQ ID NO: 34. In still other aspects of thisembodiment, a FGF-2 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 29-67, amino acids 71-111, or aminoacids 115-155 of SEQ ID NO: 34. In other aspects of this embodiment, aFGF-2 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 29-67, amino acids 71-111, or amino acids115-155 of SEQ ID NO: 34.

In another embodiment, a β-trefoil domain derived from a FGF-2 comprisesa β4/β5 hairpin turn of a β-trefoil domain of a FGF-2 or a β8/β9 hairpinturn of a β-trefoil domain of a FGF-2 of SEQ ID NO: 34. In anotheraspect of this embodiment, a β-trefoil domain derived from a FGF-2comprises amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 34.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-2 comprises a modified β4/β5 hairpin turn of aβ-trefoil domain of a FGF-2 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a FGF-2 of SEQ ID NO: 34. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-2 comprises a modification of amino acids 68-70 or aminoacids 112-114 of SEQ ID NO: 34.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-2 comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 68-70 or aminoacids 112-114 of SEQ ID NO: 34, at least 75% amino acid identity withamino acids 68-70 or amino acids 112-114 of SEQ ID NO: 34, at least 80%amino acid identity with amino acids 68-70 or amino acids 112-114 of SEQID NO: 34, at least 85% amino acid identity with amino acids 68-70 oramino acids 112-114 of SEQ ID NO: 34, at least 90% amino acid identitywith amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 34 or atleast 95% amino acid identity with amino acids 68-70 or amino acids112-114 of SEQ ID NO: 34. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-2comprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 34, at most75% amino acid identity with amino acids 68-70 or amino acids 112-114 ofSEQ ID NO: 34, at most 80% amino acid identity with amino acids 68-70 oramino acids 112-114 of SEQ ID NO: 34, at most 85% amino acid identitywith amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 34, at most90% amino acid identity with amino acids 68-70 or amino acids 112-114 ofSEQ ID NO: 34 or at most 95% amino acid identity with amino acids 68-70or amino acids 112-114 of SEQ ID NO: 34.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-2 comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 68-70 or amino acids 112-114 ofSEQ ID NO: 34. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-2 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 68-70 oramino acids 112-114 of SEQ ID NO: 34. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 34 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 34 can bereplaced with phenylalanine. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-2comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 68-70 oramino acids 112-114 of SEQ ID NO: 34. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-2 comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 68-70 or amino acids 112-114 of SEQ ID NO: 34. In still otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a FGF-2 comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid additionsrelative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 34.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-2 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 68-70 or amino acids 112-114 of SEQ IDNO: 34.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-2 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acidsubstitutions relative to amino acids 68-70 or amino acids 112-114 ofSEQ ID NO: 34. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-2 comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid substitutions relative to amino acids 68-70 or amino acids112-114 of SEQ ID NO: 34. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids68-70 or amino acids 112-114 of SEQ ID NO: 34 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 68-70 or aminoacids 112-114 of SEQ ID NO: 34 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-2 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 34.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-2 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acid deletionsrelative to amino acids 68-70 or amino acids 112-114 of SEQ ID NO: 34.In still other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-2 comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 68-70 or amino acids 112-114 of SEQ IDNO: 34. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-2 comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 68-70 or amino acids 112-114 of SEQ IDNO: 34.

In another embodiment, a β-trefoil domain derived from a FGF-4 comprisesa β-trefoil domain derived from a FGF-4 of SEQ ID NO: 35. In anotherembodiment, a β-trefoil domain derived from a FGF-4 comprises aminoacids 83-206 of SEQ ID NO: 35. In another aspect of this embodiment, aβ-trefoil domain derived from a FGF-4 comprises a α-fold motif of aβ-trefoil domain of a FGF-4, a β-fold motif of a β-trefoil domain of aFGF-4 or a γ-fold motif of a β-trefoil domain of a FGF-4 of SEQ ID NO:35. In another aspect of this embodiment, a β-trefoil domain derivedfrom a FGF-4 comprises amino acids 83-121, amino acids 125-162, or aminoacids 166-206 of SEQ ID NO: 35.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-4 comprises a β-trefoil domain with enhanced bindingactivity derived from a FGF-4 of SEQ ID NO: 35. In another embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-4comprises amino acids 83-206 of SEQ ID NO: 35. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-4 comprises a modified α-fold motif of a β-trefoil domain ofa FGF-4, a modified β-fold motif of a β-trefoil domain of a FGF-4 or amodified γ-fold motif of a β-trefoil domain of a FGF-4 of SEQ ID NO: 35.In another aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-4 comprises amino acids 83-121,amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 35.

In other aspects of this embodiment, a FGF-4 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 83-121, aminoacids 125-162, or amino acids 166-206 of SEQ ID NO: 35, at least 75%amino acid identity with amino acids 83-121, amino acids 125-162, oramino acids 166-206 of SEQ ID NO: 35, at least 80% amino acid identitywith amino acids 83-121, amino acids 125-162, or amino acids 166-206 ofSEQ ID NO: 35, at least 85% amino acid identity with amino acids 83-121,amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 35, at least90% amino acid identity with amino acids 83-121, amino acids 125-162, oramino acids 166-206 of SEQ ID NO: 35 or at least 95% amino acid identitywith amino acids 83-121, amino acids 125-162, or amino acids 166-206 ofSEQ ID NO: 35. In yet other aspects of this embodiment, a FGF-4comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO:35, at most 75% amino acid identity with amino acids 83-121, amino acids125-162, or amino acids 166-206 of SEQ ID NO: 35, at most 80% amino acididentity with amino acids 83-121, amino acids 125-162, or amino acids166-206 of SEQ ID NO: 35, at most 85% amino acid identity with aminoacids 83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO:35, at most 90% amino acid identity with amino acids 83-121, amino acids125-162, or amino acids 166-206 of SEQ ID NO: 35 or at most 95% aminoacid identity with amino acids 83-121, amino acids 125-162, or aminoacids 166-206 of SEQ ID NO: 35.

In other aspects of this embodiment, a FGF-4 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid substitutions relative to amino acids83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 35. Inother aspects of this embodiment, a FGF-4 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 83-121,amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 35. In yetother aspects of this embodiment, a FGF-4 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 83-121,amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 35. In otheraspects of this embodiment, a FGF-4 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 83-121,amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 35. In stillother aspects of this embodiment, a FGF-4 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 83-121,amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 35. In otheraspects of this embodiment, a FGF-4 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 83-121,amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 35.

In other aspects of this embodiment, a FGF-4 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid substitutions relative to amino acids83-121, amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 35. Inother aspects of this embodiment, a FGF-4 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 83-121,amino acids 125-162, or amino acids 166-206 of SEQ ID NO: 35. In yetother aspects of this embodiment, a FGF-4 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 83-121, aminoacids 125-162, or amino acids 166-206 of SEQ ID NO: 35. In other aspectsof this embodiment, a FGF-4 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 83-121, amino acids125-162, or amino acids 166-206 of SEQ ID NO: 35. In still other aspectsof this embodiment, a FGF-4 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 83-121, amino acids 125-162, oramino acids 166-206 of SEQ ID NO: 35. In other aspects of thisembodiment, a FGF-4 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 83-121, amino acids 125-162, or aminoacids 166-206 of SEQ ID NO: 35.

In another embodiment, a β-trefoil domain derived from a FGF-4 comprisesa β4/β5 hairpin turn of a β-trefoil domain of a FGF-4 or a β8/β9 hairpinturn of a β-trefoil domain of a FGF-4 of SEQ ID NO: 35. In anotheraspect of this embodiment, a β-trefoil domain derived from a FGF-4comprises amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 35.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-4 comprises a modified β4/β5 hairpin turn of aβ-trefoil domain of a FGF-4 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a FGF-4 of SEQ ID NO: 35. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-4 comprises a modification of amino acids 122-124 or aminoacids 163-165 of SEQ ID NO: 35.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-4 comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 122-124 or aminoacids 163-165 of SEQ ID NO: 35, at least 75% amino acid identity withamino acids 122-124 or amino acids 163-165 of SEQ ID NO: 35, at least80% amino acid identity with amino acids 122-124 or amino acids 163-165of SEQ ID NO: 35, at least 85% amino acid identity with amino acids122-124 or amino acids 163-165 of SEQ ID NO: 35, at least 90% amino acididentity with amino acids 122-124 or amino acids 163-165 of SEQ ID NO:35 or at least 95% amino acid identity with amino acids 122-124 or aminoacids 163-165 of SEQ ID NO: 35. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from a FGF-4comprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 35, atmost 75% amino acid identity with amino acids 122-124 or amino acids163-165 of SEQ ID NO: 35, at most 80% amino acid identity with aminoacids 122-124 or amino acids 163-165 of SEQ ID NO: 35, at most 85% aminoacid identity with amino acids 122-124 or amino acids 163-165 of SEQ IDNO: 35, at most 90% amino acid identity with amino acids 122-124 oramino acids 163-165 of SEQ ID NO: 35 or at most 95% amino acid identitywith amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 35.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-4 comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 122-124 or amino acids 163-165 ofSEQ ID NO: 35. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-4 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 122-124or amino acids 163-165 of SEQ ID NO: 35. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 35 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 35 can bereplaced with phenylalanine. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-4comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 122-124 oramino acids 163-165 of SEQ ID NO: 35. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-4 comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 122-124 or amino acids 163-165 of SEQ ID NO: 35. In still otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a FGF-4 comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid additionsrelative to amino acids 122-124 or amino acids 163-165 of SEQ ID NO: 35.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-4 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 122-124 or amino acids 163-165 of SEQID NO: 35.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-4 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acidsubstitutions relative to amino acids 122-124 or amino acids 163-165 ofSEQ ID NO: 35. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-4 comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid substitutions relative to amino acids 122-124 or amino acids163-165 of SEQ ID NO: 35. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids122-124 or amino acids 163-165 of SEQ ID NO: 35 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 122-124 oramino acids 163-165 of SEQ ID NO: 35 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-4 comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 122-124 or amino acids 163-165 of SEQID NO: 35. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-4 comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 122-124 or amino acids 163-165 of SEQID NO: 35. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-4 comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid additions relative to amino acids 122-124 or amino acids163-165 of SEQ ID NO: 35. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-4comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 122-124 or aminoacids 163-165 of SEQ ID NO: 35.

In another embodiment, a β-trefoil domain derived from a FGF-8 comprisesa β-trefoil domain derived from a FGF-8 of SEQ ID NO: 36. In anotherembodiment, a β-trefoil domain derived from a FGF-8 comprises aminoacids 43-172 of SEQ ID NO: 36. In another aspect of this embodiment, aβ-trefoil domain derived from a FGF-8 comprises a α-fold motif of aβ-trefoil domain of a FGF-8, a β-fold motif of a β-trefoil domain of aFGF-8 or a γ-fold motif of a β-trefoil domain of a FGF-8 of SEQ ID NO:36. In another aspect of this embodiment, a β-trefoil domain derivedfrom a FGF-8 comprises amino acids 43-80, amino acids 84-123, or aminoacids 127-172 of SEQ ID NO: 36.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-8 comprises a β-trefoil domain with enhanced bindingactivity derived from a FGF-8 of SEQ ID NO: 36. In another embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-8comprises amino acids 43-172 of SEQ ID NO: 36. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-8 comprises a modified α-fold motif of a β-trefoil domain ofa FGF-8, a modified β-fold motif of a β-trefoil domain of a FGF-8 or amodified γ-fold motif of a β-trefoil domain of a FGF-8 of SEQ ID NO: 36.In another aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-8 comprises amino acids 43-80, aminoacids 84-123, or amino acids 127-172 of SEQ ID NO: 36.

In other aspects of this embodiment, a FGF-8 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 43-80, aminoacids 84-123, or amino acids 127-172 of SEQ ID NO: 36, at least 75%amino acid identity with amino acids 43-80, amino acids 84-123, or aminoacids 127-172 of SEQ ID NO: 36, at least 80% amino acid identity withamino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ IDNO: 36, at least 85% amino acid identity with amino acids 43-80, aminoacids 84-123, or amino acids 127-172 of SEQ ID NO: 36, at least 90%amino acid identity with amino acids 43-80, amino acids 84-123, or aminoacids 127-172 of SEQ ID NO: 36 or at least 95% amino acid identity withamino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ IDNO: 36. In yet other aspects of this embodiment, a FGF-8 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 43-80,amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 36, at most 75%amino acid identity with amino acids 43-80, amino acids 84-123, or aminoacids 127-172 of SEQ ID NO: 36, at most 80% amino acid identity withamino acids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ IDNO: 36, at most 85% amino acid identity with amino acids 43-80, aminoacids 84-123, or amino acids 127-172 of SEQ ID NO: 36, at most 90% aminoacid identity with amino acids 43-80, amino acids 84-123, or amino acids127-172 of SEQ ID NO: 36 or at most 95% amino acid identity with aminoacids 43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO:36.

In other aspects of this embodiment, a FGF-8 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid substitutions relative to amino acids43-80, amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 36. Inother aspects of this embodiment, a FGF-8 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 43-80,amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 36. In yetother aspects of this embodiment, a FGF-8 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 43-80, aminoacids 84-123, or amino acids 127-172 of SEQ ID NO: 36. In other aspectsof this embodiment, a FGF-8 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 43-80, amino acids 84-123,or amino acids 127-172 of SEQ ID NO: 36. In still other aspects of thisembodiment, a FGF-8 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 43-80, amino acids 84-123, or aminoacids 127-172 of SEQ ID NO: 36. In other aspects of this embodiment, aFGF-8 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 43-80, amino acids 84-123, or aminoacids 127-172 of SEQ ID NO: 36.

In other aspects of this embodiment, a FGF-8 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid substitutions relative to amino acids 43-80,amino acids 84-123, or amino acids 127-172 of SEQ ID NO: 36. In otheraspects of this embodiment, a FGF-8 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 43-80, aminoacids 84-123, or amino acids 127-172 of SEQ ID NO: 36. In yet otheraspects of this embodiment, a FGF-8 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 43-80, aminoacids 84-123, or amino acids 127-172 of SEQ ID NO: 36. In other aspectsof this embodiment, a FGF-8 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 43-80, amino acids 84-123,or amino acids 127-172 of SEQ ID NO: 36. In still other aspects of thisembodiment, a FGF-8 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 43-80, amino acids 84-123, or aminoacids 127-172 of SEQ ID NO: 36. In other aspects of this embodiment, aFGF-8 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 43-80, amino acids 84-123, or amino acids127-172 of SEQ ID NO: 36.

In another embodiment, a β-trefoil domain derived from a FGF-8 comprisesa β4/β5 hairpin turn of a β-trefoil domain of a FGF-8 or a β8/β9 hairpinturn of a β-trefoil domain of a FGF-8 of SEQ ID NO: 36. In anotheraspect of this embodiment, a β-trefoil domain derived from a FGF-8comprises amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 36.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-8 comprises a modified β4/β5 hairpin turn of aβ-trefoil domain of a FGF-8 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a FGF-8 of SEQ ID NO: 36. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-8 comprises a modification of amino acids 81-83 or aminoacids 124-126 of SEQ ID NO: 36.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-8 comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 81-83 or aminoacids 124-126 of SEQ ID NO: 36, at least 75% amino acid identity withamino acids 81-83 or amino acids 124-126 of SEQ ID NO: 36, at least 80%amino acid identity with amino acids 81-83 or amino acids 124-126 of SEQID NO: 36, at least 85% amino acid identity with amino acids 81-83 oramino acids 124-126 of SEQ ID NO: 36, at least 90% amino acid identitywith amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 36 or atleast 95% amino acid identity with amino acids 81-83 or amino acids124-126 of SEQ ID NO: 36. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-8comprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 36, at most75% amino acid identity with amino acids 81-83 or amino acids 124-126 ofSEQ ID NO: 36, at most 80% amino acid identity with amino acids 81-83 oramino acids 124-126 of SEQ ID NO: 36, at most 85% amino acid identitywith amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 36, at most90% amino acid identity with amino acids 81-83 or amino acids 124-126 ofSEQ ID NO: 36 or at most 95% amino acid identity with amino acids 81-83or amino acids 124-126 of SEQ ID NO: 36.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-8 comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 81-83 or amino acids 124-126 ofSEQ ID NO: 36. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-8 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 81-83 oramino acids 124-126 of SEQ ID NO: 36. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 36 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 36 can bereplaced with phenylalanine. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-8comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 81-83 oramino acids 124-126 of SEQ ID NO: 36. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-8 comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 81-83 or amino acids 124-126 of SEQ ID NO: 36. In still otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a FGF-8 comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid additionsrelative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 36.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-8 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 81-83 or amino acids 124-126 of SEQ IDNO: 36.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-8 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acidsubstitutions relative to amino acids 81-83 or amino acids 124-126 ofSEQ ID NO: 36. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-8 comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid substitutions relative to amino acids 81-83 or amino acids124-126 of SEQ ID NO: 36. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids81-83 or amino acids 124-126 of SEQ ID NO: 36 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 81-83 or aminoacids 124-126 of SEQ ID NO: 36 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-8 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 36.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-8 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acid deletionsrelative to amino acids 81-83 or amino acids 124-126 of SEQ ID NO: 36.In still other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-8 comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 81-83 or amino acids 124-126 of SEQ IDNO: 36. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-8 comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 81-83 or amino acids 124-126 of SEQ IDNO: 36.

In another embodiment, a β-trefoil domain derived from a FGF-9 comprisesa β-trefoil domain derived from a FGF-9 of SEQ ID NO: 37. In anotherembodiment, a β-trefoil domain derived from a FGF-9 comprises aminoacids 63-196 of SEQ ID NO: 37. In another aspect of this embodiment, aβ-trefoil domain derived from a FGF-9 comprises a α-fold motif of aβ-trefoil domain of a FGF-9, a β-fold motif of a β-trefoil domain of aFGF-9 or a γ-fold motif of a β-trefoil domain of a FGF-9 of SEQ ID NO:37. In another aspect of this embodiment, a β-trefoil domain derivedfrom a FGF-9 comprises amino acids 63-100, amino acids 104-144, or aminoacids 148-196 of SEQ ID NO: 37.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-9 comprises a β-trefoil domain with enhanced bindingactivity derived from a FGF-9 of SEQ ID NO: 37. In another embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-9comprises amino acids 63-196 of SEQ ID NO: 37. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-9 comprises a modified α-fold motif of a β-trefoil domain ofa FGF-9, a modified β-fold motif of a β-trefoil domain of a FGF-9 or amodified γ-fold motif of a β-trefoil domain of a FGF-9 of SEQ ID NO: 37.In another aspect of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-9 comprises amino acids 63-100,amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 37.

In other aspects of this embodiment, a FGF-9 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 63-100, aminoacids 104-144, or amino acids 148-196 of SEQ ID NO: 37, at least 75%amino acid identity with amino acids 63-100, amino acids 104-144, oramino acids 148-196 of SEQ ID NO: 37, at least 80% amino acid identitywith amino acids 63-100, amino acids 104-144, or amino acids 148-196 ofSEQ ID NO: 37, at least 85% amino acid identity with amino acids 63-100,amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 37, at least90% amino acid identity with amino acids 63-100, amino acids 104-144, oramino acids 148-196 of SEQ ID NO: 37 or at least 95% amino acid identitywith amino acids 63-100, amino acids 104-144, or amino acids 148-196 ofSEQ ID NO: 37. In yet other aspects of this embodiment, a FGF-9comprising a β-trefoil domain with enhanced binding activity comprises apolypeptide having, e.g., at most 70% amino acid identity with aminoacids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO:37, at most 75% amino acid identity with amino acids 63-100, amino acids104-144, or amino acids 148-196 of SEQ ID NO: 37, at most 80% amino acididentity with amino acids 63-100, amino acids 104-144, or amino acids148-196 of SEQ ID NO: 37, at most 85% amino acid identity with aminoacids 63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO:37, at most 90% amino acid identity with amino acids 63-100, amino acids104-144, or amino acids 148-196 of SEQ ID NO: 37 or at most 95% aminoacid identity with amino acids 63-100, amino acids 104-144, or aminoacids 148-196 of SEQ ID NO: 37.

In other aspects of this embodiment, a FGF-9 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid substitutions relative to amino acids63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 37. Inother aspects of this embodiment, a FGF-9 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 63-100,amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 37. In yetother aspects of this embodiment, a FGF-9 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 63-100,amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 37. In otheraspects of this embodiment, a FGF-9 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 63-100,amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 37. In stillother aspects of this embodiment, a FGF-9 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 63-100,amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 37. In otheraspects of this embodiment, a FGF-9 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid additions relative to amino acids 63-100,amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 37.

In other aspects of this embodiment, a FGF-9 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid substitutions relative to amino acids63-100, amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 37. Inother aspects of this embodiment, a FGF-9 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 63-100,amino acids 104-144, or amino acids 148-196 of SEQ ID NO: 37. In yetother aspects of this embodiment, a FGF-9 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 63-100, aminoacids 104-144, or amino acids 148-196 of SEQ ID NO: 37. In other aspectsof this embodiment, a FGF-9 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 63-100, amino acids104-144, or amino acids 148-196 of SEQ ID NO: 37. In still other aspectsof this embodiment, a FGF-9 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at most one, two,three, four, five, six, seven, eight, nine, 10 or 20 contiguous aminoacid additions relative to amino acids 63-100, amino acids 104-144, oramino acids 148-196 of SEQ ID NO: 37. In other aspects of thisembodiment, a FGF-9 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 63-100, amino acids 104-144, or aminoacids 148-196 of SEQ ID NO: 37.

In another embodiment, a β-trefoil domain derived from a FGF-9 comprisesa β4/β5 hairpin turn of a β-trefoil domain of a FGF-9 or a β8/β9 hairpinturn of a β-trefoil domain of a FGF-9 of SEQ ID NO: 37. In anotheraspect of this embodiment, a β-trefoil domain derived from a FGF-9comprises amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 37.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-9 comprises a modified β4/β5 hairpin turn of aβ-trefoil domain of a FGF-9 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a FGF-9 of SEQ ID NO: 37. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-9 comprises a modification of amino acids 101-103 or aminoacids 145-147 of SEQ ID NO: 37.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-9 comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 101-103 or aminoacids 145-147 of SEQ ID NO: 37, at least 75% amino acid identity withamino acids 101-103 or amino acids 145-147 of SEQ ID NO: 37, at least80% amino acid identity with amino acids 101-103 or amino acids 145-147of SEQ ID NO: 37, at least 85% amino acid identity with amino acids101-103 or amino acids 145-147 of SEQ ID NO: 37, at least 90% amino acididentity with amino acids 101-103 or amino acids 145-147 of SEQ ID NO:37 or at least 95% amino acid identity with amino acids 101-103 or aminoacids 145-147 of SEQ ID NO: 37. In yet other aspects of this embodiment,a β-trefoil domain with enhanced binding activity derived from a FGF-9comprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 37, atmost 75% amino acid identity with amino acids 101-103 or amino acids145-147 of SEQ ID NO: 37, at most 80% amino acid identity with aminoacids 101-103 or amino acids 145-147 of SEQ ID NO: 37, at most 85% aminoacid identity with amino acids 101-103 or amino acids 145-147 of SEQ IDNO: 37, at most 90% amino acid identity with amino acids 101-103 oramino acids 145-147 of SEQ ID NO: 37 or at most 95% amino acid identitywith amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 37.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-9 comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 101-103 or amino acids 145-147 ofSEQ ID NO: 37. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-9 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 101-103or amino acids 145-147 of SEQ ID NO: 37. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 37 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 37 can bereplaced with phenylalanine. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-9comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 101-103 oramino acids 145-147 of SEQ ID NO: 37. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-9 comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 101-103 or amino acids 145-147 of SEQ ID NO: 37. In still otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a FGF-9 comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid additionsrelative to amino acids 101-103 or amino acids 145-147 of SEQ ID NO: 37.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-9 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 101-103 or amino acids 145-147 of SEQID NO: 37.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-9 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acidsubstitutions relative to amino acids 101-103 or amino acids 145-147 ofSEQ ID NO: 37. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-9 comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid substitutions relative to amino acids 101-103 or amino acids145-147 of SEQ ID NO: 37. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids101-103 or amino acids 145-147 of SEQ ID NO: 37 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 101-103 oramino acids 145-147 of SEQ ID NO: 37 can be replaced with phenylalanine.In yet other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-9 comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino aciddeletions relative to amino acids 101-103 or amino acids 145-147 of SEQID NO: 37. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-9 comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino aciddeletions relative to amino acids 101-103 or amino acids 145-147 of SEQID NO: 37. In still other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-9 comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid additions relative to amino acids 101-103 or amino acids145-147 of SEQ ID NO: 37. In other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-9comprises a polypeptide having, e.g., at least one, two, three or fourcontiguous amino acid additions relative to amino acids 101-103 or aminoacids 145-147 of SEQ ID NO: 37.

In another embodiment, a β-trefoil domain derived from a FGF-17comprises a β-trefoil domain derived from a FGF-17 of SEQ ID NO: 38. Inanother embodiment, a β-trefoil domain derived from a FGF-17 comprisesamino acids 55-183 of SEQ ID NO: 38. In another aspect of thisembodiment, a β-trefoil domain derived from a FGF-17 comprises a α-foldmotif of a β-trefoil domain of a FGF-17, a β-fold motif of a β-trefoildomain of a FGF-17 or a γ-fold motif of a β-trefoil domain of a FGF-17of SEQ ID NO: 38. In another aspect of this embodiment, a β-trefoildomain derived from a FGF-17 comprises amino acids 55-91, amino acids95-134, or amino acids 138-183 of SEQ ID NO: 38.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-17 comprises a β-trefoil domain with enhanced bindingactivity derived from a FGF-17 of SEQ ID NO: 38. In another embodiment,a β-trefoil domain with enhanced binding activity derived from a FGF-17comprises amino acids 55-183 of SEQ ID NO: 38. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-17 comprises a modified α-fold motif of a β-trefoil domain ofa FGF-17, a modified β-fold motif of a β-trefoil domain of a FGF-17 or amodified γ-fold motif of a β-trefoil domain of a FGF-17 of SEQ ID NO:38. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-17 comprises amino acids55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 38.

In other aspects of this embodiment, a FGF-17 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 38, at least 75%amino acid identity with amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 38, at least 80% amino acid identity withamino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ IDNO: 38, at least 85% amino acid identity with amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 38, at least 90%amino acid identity with amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 38 or at least 95% amino acid identity withamino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ IDNO: 38. In yet other aspects of this embodiment, a FGF-17 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 55-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 38, at most 75%amino acid identity with amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 38, at most 80% amino acid identity withamino acids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ IDNO: 38, at most 85% amino acid identity with amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 38, at most 90% aminoacid identity with amino acids 55-91, amino acids 95-134, or amino acids138-183 of SEQ ID NO: 38 or at most 95% amino acid identity with aminoacids 55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO:38.

In other aspects of this embodiment, a FGF-17 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid substitutions relative to amino acids55-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 38. Inother aspects of this embodiment, a FGF-17 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 55-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 38. In yetother aspects of this embodiment, a FGF-17 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 38. In other aspectsof this embodiment, a FGF-17 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 55-91, amino acids 95-134,or amino acids 138-183 of SEQ ID NO: 38. In still other aspects of thisembodiment, a FGF-17 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 38. In other aspects of this embodiment, aFGF-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 38.

In other aspects of this embodiment, a FGF-17 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid substitutions relative to amino acids 55-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 38. In otheraspects of this embodiment, a FGF-17 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 38. In yet otheraspects of this embodiment, a FGF-17 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 55-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 38. In other aspectsof this embodiment, a FGF-17 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 55-91, amino acids 95-134,or amino acids 138-183 of SEQ ID NO: 38. In still other aspects of thisembodiment, a FGF-17 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 55-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 38. In other aspects of this embodiment, aFGF-17 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 55-91, amino acids 95-134, or amino acids138-183 of SEQ ID NO: 38.

In another embodiment, a β-trefoil domain derived from a FGF-17comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-17 or aβ8/β9 hairpin turn of a β-trefoil domain of a FGF-17 of SEQ ID NO: 38.In another aspect of this embodiment, a β-trefoil domain derived from aFGF-17 comprises amino acids 92-94 or amino acids 135-137 of SEQ ID NO:38.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-17 comprises a modified β4/β5 hairpin turn of aβ-trefoil domain of a FGF-17 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a FGF-17 of SEQ ID NO: 38. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-17 comprises a modification of amino acids 92-94 or aminoacids 135-137 of SEQ ID NO: 38.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-17 comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 92-94 or aminoacids 135-137 of SEQ ID NO: 38, at least 75% amino acid identity withamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 38, at least 80%amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQID NO: 38, at least 85% amino acid identity with amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 38, at least 90% amino acid identitywith amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 38 or atleast 95% amino acid identity with amino acids 92-94 or amino acids135-137 of SEQ ID NO: 38. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-17comprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 38, at most75% amino acid identity with amino acids 92-94 or amino acids 135-137 ofSEQ ID NO: 38, at most 80% amino acid identity with amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 38, at most 85% amino acid identitywith amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 38, at most90% amino acid identity with amino acids 92-94 or amino acids 135-137 ofSEQ ID NO: 38 or at most 95% amino acid identity with amino acids 92-94or amino acids 135-137 of SEQ ID NO: 38.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-17 comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 92-94 or amino acids 135-137 ofSEQ ID NO: 38. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-17 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 38. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 38 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 38 can bereplaced with phenylalanine. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-17comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 38. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-17 comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 92-94 or amino acids 135-137 of SEQ ID NO: 38. In still otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a FGF-17 comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid additionsrelative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 38.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-17 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 92-94 or amino acids 135-137 of SEQ IDNO: 38.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-17 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acidsubstitutions relative to amino acids 92-94 or amino acids 135-137 ofSEQ ID NO: 38. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-17 comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid substitutions relative to amino acids 92-94 or amino acids135-137 of SEQ ID NO: 38. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids92-94 or amino acids 135-137 of SEQ ID NO: 38 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 92-94 or aminoacids 135-137 of SEQ ID NO: 38 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-17 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 38.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-17 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acid deletionsrelative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 38.In still other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-17 comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 92-94 or amino acids 135-137 of SEQ IDNO: 38. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-17 comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 92-94 or amino acids 135-137 of SEQ IDNO: 38.

In another embodiment, a β-trefoil domain derived from a FGF-18comprises a β-trefoil domain derived from a FGF-18 of SEQ ID NO: 39. Inanother embodiment, a β-trefoil domain derived from a FGF-18 comprisesamino acids 54-183 of SEQ ID NO: 39. In another aspect of thisembodiment, a β-trefoil domain derived from a FGF-18 comprises a α-foldmotif of a β-trefoil domain of a FGF-18, a β-fold motif of a β-trefoildomain of a FGF-18 or a γ-fold motif of a β-trefoil domain of a FGF-18of SEQ ID NO: 39. In another aspect of this embodiment, a β-trefoildomain derived from a FGF-18 comprises amino acids 54-91, amino acids95-134, or amino acids 138-183 of SEQ ID NO: 39.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-18 comprises a β-trefoil domain with enhanced bindingactivity derived from a FGF-18 of SEQ ID NO: 39. In another embodiment,a β-trefoil domain with enhanced binding activity derived from a FGF-18comprises amino acids 54-183 of SEQ ID NO: 39. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-18 comprises a modified α-fold motif of a β-trefoil domain ofa FGF-18, a modified β-fold motif of a β-trefoil domain of a FGF-18 or amodified γ-fold motif of a β-trefoil domain of a FGF-18 of SEQ ID NO:39. In another aspect of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-18 comprises amino acids54-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 39.

In other aspects of this embodiment, a FGF-18 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 54-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 39, at least 75%amino acid identity with amino acids 54-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 39, at least 80% amino acid identity withamino acids 54-91, amino acids 95-134, or amino acids 138-183 of SEQ IDNO: 39, at least 85% amino acid identity with amino acids 54-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 39, at least 90%amino acid identity with amino acids 54-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 39 or at least 95% amino acid identity withamino acids 54-91, amino acids 95-134, or amino acids 138-183 of SEQ IDNO: 39. In yet other aspects of this embodiment, a FGF-18 comprising aβ-trefoil domain with enhanced binding activity comprises a polypeptidehaving, e.g., at most 70% amino acid identity with amino acids 54-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 39, at most 75%amino acid identity with amino acids 54-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 39, at most 80% amino acid identity withamino acids 54-91, amino acids 95-134, or amino acids 138-183 of SEQ IDNO: 39, at most 85% amino acid identity with amino acids 54-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 39, at most 90% aminoacid identity with amino acids 54-91, amino acids 95-134, or amino acids138-183 of SEQ ID NO: 39 or at most 95% amino acid identity with aminoacids 54-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO:39.

In other aspects of this embodiment, a FGF-18 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 non-contiguous amino acid substitutions relative to amino acids54-91, amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 39. Inother aspects of this embodiment, a FGF-18 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atleast one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid substitutions relative to amino acids 54-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 39. In yetother aspects of this embodiment, a FGF-18 comprising a β-trefoil domainwith enhanced binding activity comprises a polypeptide having, e.g., atmost one, two, three, four, five, six, seven, eight, nine, 10 or 20non-contiguous amino acid deletions relative to amino acids 54-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 39. In other aspectsof this embodiment, a FGF-18 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 non-contiguousamino acid deletions relative to amino acids 54-91, amino acids 95-134,or amino acids 138-183 of SEQ ID NO: 39. In still other aspects of thisembodiment, a FGF-18 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 54-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 39. In other aspects of this embodiment, aFGF-18 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 non-contiguous amino acidadditions relative to amino acids 54-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 39.

In other aspects of this embodiment, a FGF-18 comprising a β-trefoildomain with enhanced binding activity comprises a polypeptide having,e.g., at most one, two, three, four, five, six, seven, eight, nine, 10or 20 contiguous amino acid substitutions relative to amino acids 54-91,amino acids 95-134, or amino acids 138-183 of SEQ ID NO: 39. In otheraspects of this embodiment, a FGF-18 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid substitutions relative to amino acids 54-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 39. In yet otheraspects of this embodiment, a FGF-18 comprising a β-trefoil domain withenhanced binding activity comprises a polypeptide having, e.g., at mostone, two, three, four, five, six, seven, eight, nine, 10 or 20contiguous amino acid deletions relative to amino acids 54-91, aminoacids 95-134, or amino acids 138-183 of SEQ ID NO: 39. In other aspectsof this embodiment, a FGF-18 comprising a β-trefoil domain with enhancedbinding activity comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10 or 20 contiguousamino acid deletions relative to amino acids 54-91, amino acids 95-134,or amino acids 138-183 of SEQ ID NO: 39. In still other aspects of thisembodiment, a FGF-18 comprising a β-trefoil domain with enhanced bindingactivity comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10 or 20 contiguous amino acidadditions relative to amino acids 54-91, amino acids 95-134, or aminoacids 138-183 of SEQ ID NO: 39. In other aspects of this embodiment, aFGF-18 comprising a β-trefoil domain with enhanced binding activitycomprises a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10 or 20 contiguous amino acid additionsrelative to amino acids 54-91, amino acids 95-134, or amino acids138-183 of SEQ ID NO: 39.

In another embodiment, a β-trefoil domain derived from a FGF-18comprises a β4/β5 hairpin turn of a β-trefoil domain of a FGF-18 or aβ8/β9 hairpin turn of a β-trefoil domain of a FGF-18 of SEQ ID NO: 39.In another aspect of this embodiment, a β-trefoil domain derived from aFGF-18 comprises amino acids 92-94 or amino acids 135-137 of SEQ ID NO:39.

In another embodiment, a β-trefoil domain with enhanced binding activityderived from a FGF-18 comprises a modified β4/β5 hairpin turn of aβ-trefoil domain of a FGF-18 or a modified β8/β9 hairpin turn of aβ-trefoil domain of a FGF-18 of SEQ ID NO: 39. In another aspect of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-18 comprises a modification of amino acids 92-94 or aminoacids 135-137 of SEQ ID NO: 39.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-18 comprises a polypeptide having,e.g., at least 70% amino acid identity with amino acids 92-94 or aminoacids 135-137 of SEQ ID NO: 39, at least 75% amino acid identity withamino acids 92-94 or amino acids 135-137 of SEQ ID NO: 39, at least 80%amino acid identity with amino acids 92-94 or amino acids 135-137 of SEQID NO: 39, at least 85% amino acid identity with amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 39, at least 90% amino acid identitywith amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 39 or atleast 95% amino acid identity with amino acids 92-94 or amino acids135-137 of SEQ ID NO: 39. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-18comprises a polypeptide having, e.g., at most 70% amino acid identitywith amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 39, at most75% amino acid identity with amino acids 92-94 or amino acids 135-137 ofSEQ ID NO: 39, at most 80% amino acid identity with amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 39, at most 85% amino acid identitywith amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 39, at most90% amino acid identity with amino acids 92-94 or amino acids 135-137 ofSEQ ID NO: 39 or at most 95% amino acid identity with amino acids 92-94or amino acids 135-137 of SEQ ID NO: 39.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-18 comprises a polypeptide having,e.g., at most one, two, three or four non-contiguous amino acidsubstitutions relative to amino acids 92-94 or amino acids 135-137 ofSEQ ID NO: 39. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-18 comprises apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 39. In other aspects of thisembodiment, a non-contiguous amino acid substitution of any amino acidfrom amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 39 can bereplaced with glycine. In other aspects of this embodiment, anon-contiguous amino acid substitution of any hydrophobic amino acidfrom amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 39 can bereplaced with phenylalanine. In yet other aspects of this embodiment, aβ-trefoil domain with enhanced binding activity derived from a FGF-18comprises a polypeptide having, e.g., at most one, two, three or fournon-contiguous amino acid deletions relative to amino acids 92-94 oramino acids 135-137 of SEQ ID NO: 39. In other aspects of thisembodiment, a β-trefoil domain with enhanced binding activity derivedfrom a FGF-18 comprises a polypeptide having, e.g., at least one, two,three or four non-contiguous amino acid deletions relative to aminoacids 92-94 or amino acids 135-137 of SEQ ID NO: 39. In still otheraspects of this embodiment, a β-trefoil domain with enhanced bindingactivity derived from a FGF-18 comprises a polypeptide having, e.g., atmost one, two, three or four non-contiguous amino acid additionsrelative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 39.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-18 comprises a polypeptide having,e.g., at least one, two, three or four non-contiguous amino acidadditions relative to amino acids 92-94 or amino acids 135-137 of SEQ IDNO: 39.

In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-18 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acidsubstitutions relative to amino acids 92-94 or amino acids 135-137 ofSEQ ID NO: 39. In other aspects of this embodiment, a β-trefoil domainwith enhanced binding activity derived from a FGF-18 comprises apolypeptide having, e.g., at least one, two, three or four contiguousamino acid substitutions relative to amino acids 92-94 or amino acids135-137 of SEQ ID NO: 39. In other aspects of this embodiment,contiguous amino acid substitutions of amino acids from amino acids92-94 or amino acids 135-137 of SEQ ID NO: 39 can be replaced withglycine. In other aspects of this embodiment, contiguous amino acidsubstitutions of hydrophobic amino acids from amino acids 92-94 or aminoacids 135-137 of SEQ ID NO: 39 can be replaced with phenylalanine. Inyet other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-18 comprises a polypeptide having,e.g., at most one, two, three or four contiguous amino acid deletionsrelative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 39.In other aspects of this embodiment, a β-trefoil domain with enhancedbinding activity derived from a FGF-18 comprises a polypeptide having,e.g., at least one, two, three or four contiguous amino acid deletionsrelative to amino acids 92-94 or amino acids 135-137 of SEQ ID NO: 39.In still other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-18 comprises a polypeptidehaving, e.g., at most one, two, three or four contiguous amino acidadditions relative to amino acids 92-94 or amino acids 135-137 of SEQ IDNO: 39. In other aspects of this embodiment, a β-trefoil domain withenhanced binding activity derived from a FGF-18 comprises a polypeptidehaving, e.g., at least one, two, three or four contiguous amino acidadditions relative to amino acids 92-94 or amino acids 135-137 of SEQ IDNO: 39.

Because of the modular nature of the β-trefoil domain, it is envisionedthat any combination of α-folds, β-folds, γ-folds, β4/β5 β-hairpinturns, β8/β9 β-hairpin turns, or any combination thereof from aβ-trefoil domain of a Clostridial toxin binding domain, a β-trefoildomain of a modified Clostridial toxin binding domain, a β-trefoildomain of a NAP or a β-trefoil domain, a β-trefoil domain of a modifiedNAP, a β-trefoil domain of an FGF ligand, a β-trefoil domain of amodified FGF ligand, or any combination thereof, can be used to practiceaspect of the present invention. As a non-limiting example, a modifiedClostridial toxin disclosed in the present specification can comprise anenhanced targeting domain comprising a α-fold from a BoNT/A bindingdomain, a β-fold from a FGF-18 and a 2γ-fold from a Clostridialbotulinum serotype A HA-33. As another non-limiting example, a modifiedClostridial toxin disclosed in the present specification can comprise anenhanced targeting domain comprising a α-fold from a Clostridialbotulinum serotype A HA-17, a β-fold from a Clostridial botulinumserotype E NTNH and a γ-fold from a modified BoNT/C1 binding domain withenhanced binding activity.

In another aspect of the invention, a modified Clostridial toxin withenhanced binding activity comprises, in part, a protease cleavage siteis located within a di-chain loop region. As used herein, the term“di-chain loop region” means the amino acid sequence of a Clostridialtoxin containing a protease cleavage site used to convert thesingle-chain form of a Clostridial toxin into the di-chain form.Non-limiting examples of a Clostridial toxin di-chain loop region,include, a di-chain loop region of BoNT/A comprising amino acids 430-454of SEQ ID NO: 1; a di-chain loop region of BoNT/B comprising amino acids437-446 of SEQ ID NO: 2; a di-chain loop region of BoNT/C1 comprisingamino acids 437-453 of SEQ ID NO: 3; a di-chain loop region of BoNT/Dcomprising amino acids 437-450 of SEQ ID NO: 4; a di-chain loop regionof BoNT/E comprising amino acids 412-426 of SEQ ID NO: 5; a di-chainloop region of BoNT/F comprising amino acids 429-445 of SEQ ID NO: 6; adi-chain loop region of BoNT/G comprising amino acids 436-450 of SEQ IDNO: 7; and a di-chain loop region of TeNT comprising amino acids 439-467of SEQ ID NO: 8 (Table 8).

TABLE 8 Di-chain Loop Region of Clostridial Toxins SEQDi-chain Loop Region Containing ID the Naturally-occurring Heavy ChainToxin NO: Light Chain Region Protease Cleavage Site Region BoNT/A 1NMNFTKLKNFTGLFEFYKLL CVRGIITSKTKSLDKGYNK*----ALNDLC IKVNNWDL BoNT/B 2KQAYEEISKEHLAVYKIQM CKSVK*-------------------APGIC IDVDNEDL BoNT/C1 3PALRKVNPENMLYLFTKF CHKAIDGRSLYNK*------------TLDC RELLVKNTDL BoNT/D 4PALQKLSSESVVDLFTKV CLRLTKNSR*---------------DDSTC IKVKNNRL BoNT/E 5IITPITGRGLVKKIIRF CKNIVSVKGIR*--------------KSIC IEINNGEL BoNT/F 6IIDSIPDKGLVEKIVKF CKSVIPRKGTK*------------APPRLC IRVNNSEL BoNT/G 7KEAYEEISLEHLVIYRIAM CKPVMYKNTGK*--------------SEQC IIVNNEDL TeNT 8TNAFRNVDGSGLVSKLIGL CKKIIPPTNIRENLYNRTA*SLTDLGGELC IKIKNEDL The aminoacid sequence displayed are as follows: BoNT/A, residues 325-462 of SEQID No: 1; BoNT/B, residues 332-454 of SEQ ID No: 2; BoNT/C1, residues334-463 of SEQ ID No: 3; BoNT/D, residues 334-458 of SEQ ID No: 4;BoNT/E, residues 311-434 of SEQ ID No: 5; BoNT/F, residues 328-453 ofSEQ ID No: 6; BoNT/G, residues 331-458 of SEQ ID No: 7; and TeNT,residues 334-474 of SEQ ID No: 8. An asterisks (*) indicates a peptidebond that is cleaved by a Clostridial toxin protease.

In is envisioned that any and all protease cleavage sites can be used toconvert the single-chain polypeptide form of a Clostridial toxin intothe di-chain form, including, without limitation, endogenous di-chainloop protease cleavage sites and exogenous protease cleavage sites. Thelocation and kind of protease cleavage site may be critical becausecertain targeting domains require a free amino-terminal orcarboxyl-terminal amino acid. For example, when a targeting domain isplaced between two other domains, e.g., see FIG. 5, a criteria forselection of a protease cleavage site could be whether the protease thatcleaves its site leaves a flush cut, exposing the free amino-terminal orcarboxyl-terminal of the altered targeting domain necessary forselective binding of the targeting domain to its receptor. The selectionand placement of a protease cleavage site is well known in the art.

As used herein, the term “endogenous di-chain loop protease cleavagesite” is synonymous with a “naturally occurring di-chain loop proteasecleavage site” and means a naturally occurring protease cleavage sitefound within the di-chain loop region of a naturally occurringClostridial toxin and includes, without limitation, naturally occurringClostridial toxin di-chain loop protease cleavage site variants, suchas, e.g., Clostridial toxin di-chain loop protease cleavage siteisoforms and Clostridial toxin di-chain loop protease cleavage sitesubtypes. Non-limiting examples of an endogenous protease cleavage site,include, e.g., a BoNT/A di-chain loop protease cleavage site, a BoNT/Bdi-chain loop protease cleavage site, a BoNT/C1 di-chain loop proteasecleavage site, a BoNT/D di-chain loop protease cleavage site, a BoNT/Edi-chain loop protease cleavage site, a BoNT/F di-chain loop proteasecleavage site, a BoNT/G di-chain loop protease cleavage site and a TeNTdi-chain loop protease cleavage site.

As mentioned above, Clostridial toxins are translated as a single-chainpolypeptide of approximately 150 kDa that is subsequently cleaved byproteolytic scission within a disulfide loop by a naturally-occurringprotease. This posttranslational processing yields a di-chain moleculecomprising an approximately 50 kDa light chain (LC) and an approximately100 kDa heavy chain (HC) held together by a single disulphide bond andnoncovalent interactions. While the identity of the protease iscurrently unknown, the di-chain loop protease cleavage site for manyClostridial toxins has been proposed. In BoNTs, cleavage at K448-A449converts the single polypeptide form of BoNT/A into the di-chain form;cleavage at K441-A442 converts the single polypeptide form of BoNT/Binto the di-chain form; cleavage at K449-T450 converts the singlepolypeptide form of BoNT/C1 into the di-chain form; cleavage atR445-D446 converts the single polypeptide form of BoNT/D into thedi-chain form; cleavage at R422-K423 converts the single polypeptideform of BoNT/E into the di-chain form; cleavage at K439-A440 convertsthe single polypeptide form of BoNT/F into the di-chain form; andcleavage at K446-S447 converts the single polypeptide form of BoNT/Ginto the di-chain form. Proteolytic cleavage of the single polypeptideform of TeNT at A457-S458 results in the di-chain form. Such a di-chainloop protease cleavage site is operably-linked in-frame to a modifiedClostridial toxin as a fusion protein.

However, it should also be noted that additional cleavage sites withinthe di-chain loop also appear to be cleaved resulting in the generationof a small peptide fragment being lost. As a non-limiting example,BoNT/A single-chain polypeptide cleave ultimately results in the loss ofa ten amino acid fragment within the di-chain loop. Thus, in BoNTs,cleavage at S441-L442 converts the single polypeptide form of BoNT/Ainto the di-chain form; cleavage at G444-I445 converts the singlepolypeptide form of BoNT/B into the di-chain form; cleavage at S445-L446converts the single polypeptide form of BoNT/C1 into the di-chain form;cleavage at K442-N443 converts the single polypeptide form of BoNT/Dinto the di-chain form; cleavage at K419-G420 converts the singlepolypeptide form of BoNT/E into the di-chain form; cleavage at K423-S424converts the single polypeptide form of BoNT/E into the di-chain form;cleavage at K436-G437 converts the single polypeptide form of BoNT/Finto the di-chain form; cleavage at T444-G445 converts the singlepolypeptide form of BoNT/G into the di-chain form; and cleavage atE448-Q449 converts the single polypeptide form of BoNT/G into thedi-chain form.

Thus, in an embodiment, a protease cleavage site comprising anendogenous Clostridial toxin di-chain loop protease cleavage site isused to convert the single-chain toxin into the di-chain form. Inaspects of this embodiment, conversion into the di-chain form byproteolytic cleavage occurs from a site comprising, e.g., a BoNT/Adi-chain loop protease cleavage site, a BoNT/B di-chain loop proteasecleavage site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/Ddi-chain loop protease cleavage site, a BoNT/E di-chain loop proteasecleavage site, a BoNT/F di-chain loop protease cleavage site, a BoNT/Gdi-chain loop protease cleavage site or a TeNT di-chain loop proteasecleavage site.

In other aspects of this embodiment, conversion into the di-chain formby proteolytic cleavage occurs from a site comprising, e.g., a di-chainloop region of BoNT/A comprising amino acids 430-454 of SEQ ID NO: 1; adi-chain loop region of BoNT/B comprising amino acids 437-446 of SEQ IDNO: 2; a di-chain loop region of BoNT/C1 comprising amino acids 437-453of SEQ ID NO: 3; a di-chain loop region of BoNT/D comprising amino acids437-450 of SEQ ID NO: 4; a di-chain loop region of BoNT/E comprisingamino acids 412-426 of SEQ ID NO: 5; a di-chain loop region of BoNT/Fcomprising amino acids 429-445 of SEQ ID NO: 6; a di-chain loop regionof BoNT/G comprising amino acids 436-450 of SEQ ID NO: 7; or a di-chainloop region of TeNT comprising amino acids 439-467 of SEQ ID NO: 8.

It is also envisioned that an exogenous protease cleavage site can beused to convert the single-chain polypeptide form of a modifiedClostridial toxin disclosed in the present specification into thedi-chain form. As used herein, the term “exogenous protease cleavagesite” is synonymous with a “non-naturally occurring protease cleavagesite” and means a protease cleavage site that is not normally present ina di-chain loop region from a naturally occurring Clostridial toxin.Non-limiting examples of exogenous protease cleavage sites include,e.g., an enterokinase cleavage site (Table 9); a Thrombin cleavage site(Table 9); a Factor Xa cleavage site (Table 9); a human rhinovirus 3Cprotease cleavage site (Table 9); a tobacco etch virus (TEV) proteasecleavage site (Table 9); a dipeptidyl aminopeptidase cleavage site; asmall ubiquitin-like modifier (SUMO)/ubiquitin-like protein-1(ULP-1)protease cleavage site, such as, e.g.,MADSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID. NO: 57); and aClostridial toxin substrate cleavage site.

TABLE 9 Exogenous Protease Cleavage Sites Protease Cleavage Non-limitingSEQ ID Site Consensus Sequence Examples NO: Bovine enterokinase DDDDK*DDDDK* 40 Tobacco Etch Virus E P⁵ P⁴YP²Q*(G/S), ENLYFQ*G 41 (TEV) whereP², P⁴ and P⁵ can be any amino acid ENLYFQ*S 42 ENIYTQ*G 43 ENIYTQ*S 44ENIYLQ*G 45 ENIYLQ*S 46 ENVYFQ*G 47 ENVYSQ*S 48 ENVYSQ*G 49 ENVYSQ*S 50Human Rhinovirus 3C P⁵P⁴LFQ*GP EALFQ*GP 51 where P⁴ is G, A, V, L, I, M,S or T and P⁵ can any EVLFQ*GP 52 amino acid, with D or E preferred.ELLFQ*GP 53 DALFQ*GP 54 DVLFQ*GP 55 DLLFQ*GP 56 SUMO/ULP-1 Tertiarystructure polypeptide-G* 57 Thrombin P³P²(R/K)*P^(1′), GVR*G 58 where P³is any amino acid and P² or P^(1′) is G with SAR*G 59 the other positionbeing any amino acid SLR*G 60 DGR*I 61 QGK*I 62 ThrombinP⁴P³P(R/K)*P^(1′)P^(2′) LVPR*GS 63 where P^(1′) and P^(2′) can be anyamino acid except for LVPK*GS 64 acidic amino acids like D or E; and P³and P⁴ are FIPR*TF 65 hydrophobic amino acids like F, L, I, Y, W, V, M,P, VLPR*SF 66 C or A IVPR*SF 67 IVPR*GY 68 VVPR*GV 69 VLPR*LI 70 VMPR*SL71 MFPR*SL 72 Coagulation Factor Xa I(E/D)GR* IDGR* 73 IEGR* 74 Anasterisks (*) indicates the peptide bond that is cleaved by theindicated protease.

As mentioned above, a Clostridial toxin is converted from a singlepolypeptide form into a di-chain molecule by proteolytic cleavage. Whilethe naturally-occurring protease is currently not known, cleavage occurswithin the di-chain loop region between the two cysteine residues thatform the disulfide bridge (see Table 8). Replacement of an endogenousprotease cleavage site with an exogenous protease cleavage site willenable cleavage of a modified Clostridial toxin disclosed in the presentspecification when expressed in an organism that does not produce thenaturally-occurring protease used to cleave the di-chain loop region ofa toxin. Similarly, an addition of an exogenous protease cleavage sitein the di-chain loop region will also enable cleavage of a modifiedClostridial toxin disclosed in the present specification when expressedin an organism that does not produce the naturally-occurring proteaseused to cleave the di-chain loop region of a toxin.

It is envisioned that an exogenous protease cleavage site of any and alllengths can be useful in aspects of the present invention with theproviso that the exogenous protease cleavage site is capable of beingcleaved by its respective protease. Thus, in aspects of this embodiment,an exogenous protease cleavage site can be, e.g., at least 6 amino acidsin length, at least 7 amino acids in length, at least 8 amino acids inlength, at least 9 amino acids in length, at least 10 amino acids inlength, at least 15 amino acids in length, at least 20 amino acids inlength, at least 25 amino acids in length, at least 30 amino acids inlength, at least 40 amino acids in length, at least 50 amino acids inlength or at least 60 amino acids in length. In other aspects of thisembodiment, an exogenous protease cleavage site can be, e.g., at most 6amino acids in length, at most 7 amino acids in length, at most 8 aminoacids in length, at most 9 amino acids in length, at most 10 amino acidsin length, at most 15 amino acids in length, at most 20 amino acids inlength, at most 25 amino acids in length, at most 30 amino acids inlength, at most 40 amino acids in length, at most 50 amino acids inlength or at most 60 amino acids in length.

In aspects of this embodiment, a di-chain loop region can be modified tosubstitute a naturally-occurring protease cleavage site for an exogenousprotease cleavage site. In this type of modification, thenaturally-occurring protease cleavage site is made inoperable and thuscan not be cleaved by its protease. Only the exogenous protease cleavagesite can be cleaved by its corresponding exogenous protease. In thistype of modification, the exogenous protease site is operably-linkedin-frame to a modified Clostridial toxin as a fusion protein and thesite can be cleaved by its respective exogenous protease. As anon-limiting example, a single-chain modified BoNT/A comprising anexogenous protease cleavage site in the di-chain loop region can becleaved by its respective exogenous protease to produce the di-chainform of the toxin.

In other aspects of this embodiment, a di-chain loop region can bemodified to include an exogenous protease cleavage site in addition tothe naturally-occurring protease cleavage site. In this type ofmodification, both cleavage sites are operably-linked in-frame to amodified Clostridial toxin as a fusion protein and both sites can becleaved by their respective proteases. As a non-limiting example, asingle-chain modified BoNT/A that comprises a di-chain loop containingboth the naturally-occurring BoNT/A di-chain loop protease cleavage siteand an exogenous protease cleavage site can be cleaved by either thenaturally occurring di-chain loop protease or by the appropriateexogenous protease to produce the di-chain form of the toxin.

A naturally-occurring protease cleavage site can be made inoperable byaltering at least the two amino acids flanking the peptide bond cleavedby the naturally-occurring di-chain loop protease. More extensivealterations can be made, with the proviso that the two cysteine residuesof the di-chain loop region remain intact and can still form thedisulfide bridge. Non-limiting examples of an amino acid alterationinclude deletion of an amino acid or replacement of the original aminoacid with a different amino acid. Thus, in one embodiment, anaturally-occurring protease cleavage site is made inoperable byaltering the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease. In other aspects of this embodiment, anaturally-occurring protease cleavage site is made inoperable byaltering, e.g., at least three amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease; atleast four amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at least fiveamino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at least six amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least seven amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least eight amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least nine amino acids including thetwo amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at least ten amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; at least 15 amino acids including the two amino acids flankingthe peptide bond cleaved by a naturally-occurring protease; or at least20 amino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease.

In still other aspects of this embodiment, a naturally-occurringdi-chain protease cleavage site is made inoperable by altering, e.g., atmost three amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at most fouramino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at most five amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at most six amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; at most seven amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease; atmost eight amino acids including the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease; at most nineamino acids including the two amino acids flanking the peptide bondcleaved by a naturally-occurring protease; at most ten amino acidsincluding the two amino acids flanking the peptide bond cleaved by anaturally-occurring protease; at most 15 amino acids including the twoamino acids flanking the peptide bond cleaved by a naturally-occurringprotease; or at most 20 amino acids including the two amino acidsflanking the peptide bond cleaved by a naturally-occurring protease.

In an embodiment, an exogenous protease cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In aspects of thisembodiment, a modified Clostridial toxin comprises an exogenous proteasecleavage site comprises, e.g., a bovine enterokinase protease cleavagesite, a Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3Cprotease cleavage site, a SUMO/ULP-1 protease cleavage site, a Thrombinprotease cleavage site or a Factor Xa protease cleavage site. In otheraspects of this embodiment, an exogenous protease cleavage site islocated within the di-chain loop of, e.g., a modified BoNT/A, a modifiedBoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, amodified BoNT/F, a modified BoNT/G or a modified TeNT.

In an aspect of this embodiment, an exogenous protease cleavage site canbe, e.g., a bovine enterokinase cleavage site is located within thedi-chain loop of a modified Clostridial toxin. In other aspects of theembodiment, an exogenous protease cleavage site can be, e.g., a bovineenterokinase protease cleavage site located within the di-chain loop ofa modified Clostridial toxin comprises SEQ ID NO: 40. Is still otheraspects of this embodiment, a bovine enterokinase protease cleavage siteis located within the di-chain loop of, e.g., a modified BoNT/A, amodified BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modifiedBoNT/E, a modified BoNT/F, a modified BoNT/G or a modified TeNT.

In another aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a Tobacco Etch Virus protease cleavage site islocated within the di-chain loop of a modified Clostridial toxin. Inother aspects of the embodiment, an exogenous protease cleavage site canbe, e.g., a Tobacco Etch Virus protease cleavage site located within thedi-chain loop of a modified Clostridial toxin comprises SEQ ID NO: 41,SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50. Isstill other aspects of this embodiment, a Tobacco Etch Virus proteasecleavage site is located within the di-chain loop of, e.g., a modifiedBoNT/A, a modified BoNT/B, a modified BoNT/C1, a modified BoNT/D, amodified BoNT/E, a modified BoNT/F, a modified BoNT/G or a modifiedTeNT.

In still another aspect of this embodiment, an exogenous proteasecleavage site can be, e.g., a Human Rhinovirus 3C protease cleavage siteis located within the di-chain loop of a modified Clostridial toxin. Inother aspects of the embodiment, an exogenous protease cleavage site canbe, e.g., a Human Rhinovirus 3C protease cleavage site located withinthe di-chain loop of a modified Clostridial toxin comprises SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 or SEQ IDNO: 56. Is still other aspects of this embodiment, a Human Rhinovirus 3Cprotease cleavage site is located within the di-chain loop of, e.g., amodified BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modifiedBoNT/D, a modified BoNT/E, a modified BoNT/F, a modified BoNT/G or amodified TeNT.

In yet another aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a SUMO/ULP-1 protease cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In other aspects ofthe embodiment, an exogenous protease cleavage site can be, e.g., aSUMO/ULP-1 protease cleavage site located within the di-chain loop of amodified Clostridial toxin comprises SEQ ID NO: 57. Is still otheraspects of this embodiment, a SUMO/ULP-1 protease cleavage site islocated within the di-chain loop of, e.g., a modified BoNT/A, a modifiedBoNT/B, a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, amodified BoNT/F, a modified BoNT/G or a modified TeNT.

In a further aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a Thrombin protease cleavage site is located withinthe di-chain loop of a modified Clostridial toxin. In other aspects ofthe embodiment, an exogenous protease cleavage site can be, e.g., aThrombin protease cleavage site located within the di-chain loop of amodified Clostridial toxin comprises SEQ ID NO: 58, SEQ ID NO: 59, SEQID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64,SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO: 70, SEQ ID NO: 71 or SEQ ID NO: 72. Is still otheraspects of this embodiment, a Thrombin protease cleavage site is locatedwithin the di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B,a modified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modifiedBoNT/F, a modified BoNT/G or a modified TeNT.

In another aspect of this embodiment, an exogenous protease cleavagesite can be, e.g., a Coagulation Factor Xa protease cleavage site islocated within the di-chain loop of a modified Clostridial toxin. Inother aspects of the embodiment, an exogenous protease cleavage site canbe, e.g., a Coagulation Factor Xa protease cleavage site located withinthe di-chain loop of a modified Clostridial toxin comprises SEQ ID NO:73 or SEQ ID NO: 74. Is still other aspects of this embodiment, aCoagulation Factor Xa protease cleavage site is located within thedi-chain loop of, e.g., a modified BoNT/A, a modified BoNT/B, a modifiedBoNT/C1, a modified BoNT/D, a modified BoNT/E, a modified BoNT/F, amodified BoNT/G or a modified TeNT.

In another embodiment, an exogenous protease site comprises aClostridial toxin substrate cleavage site. As used herein, the term“Clostridial toxin substrate cleavage site” means a scissile bondtogether with adjacent or non-adjacent recognition elements, or both,sufficient for detectable proteolysis at the scissile bond by aClostridial toxin under conditions suitable for Clostridial toxinprotease activity. By definition, a Clostridial toxin substrate cleavagesite is susceptible to cleavage by at least one Clostridial toxin underconditions suitable for Clostridial toxin protease activity.Non-limiting examples of Clostridial toxin substrate cleavage site aredisclosed in, e.g., Steward, L. E. et al., Self-Activating ClostridialToxins, U.S. Patent Application 60/718,616 (Sep. 19, 2005).

It is understood that a modified Clostridial toxin disclosed in thepresent specification can optionally include one or more additionalcomponents. As a non-limiting example of an optional component, amodified Clostridial toxin can further comprise a flexible regioncomprising a flexible spacer. Non-limiting examples of a flexible spacerinclude, e.g., a G-spacer GGGGS (SEQ ID NO: 75) or an A-spacer EAAAK(SEQ ID NO: 76). A flexible region comprising flexible spacers can beused to adjust the length of a polypeptide region in order to optimize acharacteristic, attribute or property of a polypeptide. Such a flexibleregion is operably-linked in-frame to the modified Clostridial toxin asa fusion protein. As a non-limiting example, a polypeptide regioncomprising one or more flexible spacers in tandem can be use to betterexpose a protease cleavage site thereby facilitating cleavage of thatsite by a protease. As another non-limiting example, a polypeptideregion comprising one or more flexible spacers in tandem can be use tobetter present an enhanced targeting domain, thereby facilitating thebinding of that enhanced targeting domain to its receptor.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprise a flexible region comprisinga flexible spacer. In another embodiment, a modified Clostridial toxindisclosed in the present specification can further comprise flexibleregion comprising a plurality of flexible spacers in tandem. In aspectsof this embodiment, a flexible region can comprise in tandem, e.g., atleast 1 G-spacer, at least 2 G-spacers, at least 3 G-spacers, at least 4G-spacers or at least 5 G-spacers. In other aspects of this embodiment,a flexible region can comprise in tandem, e.g., at most 1 G-spacer, atmost 2 G-spacers, at most 3 G-spacers, at most 4 G-spacers or at most 5G-spacers. In still other aspects of this embodiment, a flexible regioncan comprise in tandem, e.g., at least 1 A-spacer, at least 2 A-spacers,at least 3 A-spacers, at least 4 A-spacers or at least 5 A-spacers. Instill other aspects of this embodiment, a flexible region can comprisein tandem, e.g., at most 1 A-spacer, at most 2 A-spacers, at most 3A-spacers, at most 4 A-spacers or at most 5 A-spacers. In another aspectof this embodiment, a modified Clostridial toxin can comprise a flexibleregion comprising one or more copies of the same flexible spacers, oneor more copies of different flexible-spacer regions, or any combinationthereof.

In aspects of this embodiment, a modified Clostridial toxin comprising aflexible spacer can be, e.g., a modified BoNT/A, a modified BoNT/B, amodified BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modifiedBoNT/F, a modified BoNT/G or a modified TeNT.

It is envisioned that a modified Clostridial toxin disclosed in thepresent specification can comprise a flexible spacer in any and alllocations with the proviso that modified Clostridial toxin is capable ofperforming the intoxication process. In aspects of this embodiment, aflexible spacer is positioned between, e.g., an enzymatic domain and atranslocation domain, an enzymatic domain and an enhanced targetingdomain, an enzymatic domain and a protease cleavage site. In otheraspects of this embodiment, a G-spacer is positioned between, e.g., anenzymatic domain and a translocation domain, an enzymatic domain and anenhanced targeting domain, an enzymatic domain and a protease cleavagesite. In other aspects of this embodiment, a A-spacer is positionedbetween, e.g., an enzymatic domain and a translocation domain, anenzymatic domain and an enhanced targeting domain, an enzymatic domainand a protease cleavage site.

In other aspects of this embodiment, a flexible spacer is positionedbetween, e.g., an enhanced targeting domain and a translocation domain,an enhanced targeting domain and an enzymatic domain, an enhancedtargeting domain and a protease cleavage site. In other aspects of thisembodiment, a G-spacer is positioned between, e.g., an enhancedtargeting domain and a translocation domain, an enhanced targetingdomain and an enzymatic domain, an enhanced targeting domain and aprotease cleavage site. In other aspects of this embodiment, a A-spaceris positioned between, e.g., an enhanced targeting domain and atranslocation domain, an enhanced targeting domain and an enzymaticdomain, an enhanced targeting domain and a protease cleavage site.

In yet other aspects of this embodiment, a flexible spacer is positionedbetween, e.g., a translocation domain and an enzymatic domain, antranslocation domain and an enhanced targeting domain, an translocationdomain and a protease cleavage site. In other aspects of thisembodiment, a G-spacer is positioned between, e.g., a translocationdomain and an enzymatic domain, an translocation domain and an enhancedtargeting domain, an translocation domain and a protease cleavage site.In other aspects of this embodiment, a A-spacer is positioned between,e.g., a translocation domain and an enzymatic domain, an translocationdomain and an enhanced targeting domain, a translocation domain and aprotease cleavage site.

As another non-limiting example of an optional component, a modifiedClostridial toxin can further comprise an epitope-binding region. Anepitope-binding region can be used in a wide variety of proceduresinvolving, e.g., protein purification and protein visualization. Such anepitope-binding region is operably-linked in-frame to a modifiedClostridial toxin as a fusion protein. Non-limiting examples of anepitope-binding region include, e.g., FLAG, Express™ (SEQ ID NO: 77),human Influenza virus hemagluttinin (HA) (SEQ ID NO: 78), humanp62^(c-Myc) protein (c-MYC) (SEQ ID NO: 79), Vesicular Stomatitis VirusGlycoprotein (VSV-G) (SEQ ID NO: 80), Substance P (SEQ ID NO: 81),glycoprotein-D precursor of Herpes simplex virus (HSV) (SEQ ID NO: 82),V5 (SEQ ID NO: 83), AU1 (SEQ ID NO: 84) and AU5 (SEQ ID NO: 85);affinity-binding, such as. e.g., polyhistidine (HIS) (SEQ ID NO: 86),streptavidin binding peptide (strep), and biotin or a biotinylationsequence; peptide-binding regions, such as. e.g., the glutathionebinding domain of glutathione-S-transferase, the calmodulin bindingdomain of the calmodulin binding protein, and the maltose binding domainof the maltose binding protein. Non-limiting examples of specificprotocols for selecting, making and using an appropriate binding peptideare described in, e.g., Epitope Tagging, pp. 17.90-17.93 (Sambrook andRussell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed.2001); Antibodies: A Laboratory Manual (Edward Harlow & David Lane,eds., Cold Spring Harbor Laboratory Press, 2^(nd) ed. 1998); and UsingAntibodies: A Laboratory Manual: Portable Protocol No. I (Edward Harlow& David Lane, Cold Spring Harbor Laboratory Press, 1998). In addition,non-limiting examples of binding peptides as well as well-characterizedreagents, conditions and protocols are readily available from commercialvendors that include, without limitation, BD Biosciences-Clontech, PaloAlto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen,Inc, Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene,La Jolla, Calif. These protocols are routine procedures well within thescope of one skilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprise an epitope-binding region. Inanother embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprises a plurality ofepitope-binding regions. In aspects of this embodiment, a modifiedClostridial toxin can comprise, e.g., at least 1 epitope-binding region,at least 2 epitope-binding regions, at least 3 epitope-binding regions,at least 4 epitope-binding regions or at least 5 epitope-bindingregions. In other aspects of this embodiment, a modified Clostridialtoxin can comprise, e.g., at most 1 epitope-binding region, at most 2epitope-binding regions, at most 3 epitope-binding regions, at most 4epitope-binding regions or at most 5 epitope-binding regions. In anotheraspect of this embodiment, a modified Clostridial toxin can comprise oneor more copies of the same epitope-binding region, one or more copies ofdifferent epitope-binding regions, or any combination thereof.

The location of an epitope-binding region can be in various positions,including, without limitation, at the amino terminus of a modifiedClostridial toxin, within a modified Clostridial toxin, or at thecarboxyl terminus of a modified Clostridial toxin. Thus, in anembodiment, an epitope-binding region is located at the amino-terminusof a modified Clostridial toxin. In such a location, a start methionineshould be placed in front of the epitope-binding region. In addition, itis known in the art that when adding a polypeptide that isoperationally-linked to the amino terminus of another polypeptidecomprising the start methionine that the original methionine residue canbe deleted. This is due to the fact that the added polypeptide willcontain a new start methionine and that the original start methioninemay reduce optimal expression of the fusion protein. In aspects of thisembodiment, an epitope-binding region located at the amino-terminus of amodified Clostridial toxin disclosed in the present specification canbe, e.g., a FLAG, Express™ epitope-binding region, a human Influenzavirus hemagluttinin (HA) epitope-binding region, a human p62^(c-Myc)protein (c-MYC) epitope-binding region, a Vesicular Stomatitis VirusGlycoprotein (VSV-G) epitope-binding region, a Substance Pepitope-binding region, a glycoprotein-D precursor of Herpes simplexvirus (HSV) epitope-binding region, a V5 epitope-binding region, a AU1epitope-binding region, a AU5 epitope-binding region, a polyhistidineepitope-binding region, a streptavidin binding peptide epitope-bindingregion, a biotin epitope-binding region, a biotinylation epitope-bindingregion, a glutathione binding domain of glutathione-S-transferase, acalmodulin binding domain of the calmodulin binding protein or a maltosebinding domain of the maltose binding protein.

In another embodiment, an epitope-binding region is located at thecarboxyl-terminus of a modified Clostridial toxin. In aspects of thisembodiment, an epitope-binding region located at the carboxyl-terminusof a modified Clostridial toxin disclosed in the present specificationcan be, e.g., a FLAG, Express™ epitope-binding region, a human Influenzavirus hemagluttinin (HA) epitope-binding region, a human p62^(c-Myc)protein (c-MYC) epitope-binding region, a Vesicular Stomatitis VirusGlycoprotein (VSV-G) epitope-binding region, a Substance Pepitope-binding region, a glycoprotein-D precursor of Herpes simplexvirus (HSV) epitope-binding region, a V5 epitope-binding region, a AU1epitope-binding region, a AU5 epitope-binding region, a polyhistidineepitope-binding region, a streptavidin binding peptide epitope-bindingregion, a biotin epitope-binding region, a biotinylation epitope-bindingregion, a glutathione binding domain of glutathione-S-transferase, acalmodulin binding domain of the calmodulin binding protein or a maltosebinding domain of the maltose binding protein.

It is envisioned that a modified Clostridial toxin disclosed in thepresent specification can comprise an enhance targeting domain in anyand all locations with the proviso that modified Clostridial toxin iscapable of performing the intoxication process. Non-limiting examplesinclude, locating an enhance targeting domain at the amino terminus of amodified Clostridial toxin (see FIG. 4); locating an enhance targetingdomain between a Clostridial toxin enzymatic domain and a Clostridialtoxin translocation domain of a modified Clostridial toxin (see FIG. 5);and locating an enhance targeting domain at the carboxyl terminus of amodified Clostridial toxin (see FIG. 6). The enzymatic domain ofnaturally-occurring Clostridial toxins contains the native startmethionine. Thus, in domain organizations where the enzymatic domain isnot in the amino-terminal location an amino acid sequence comprising thestart methionine should be placed in front of the amino-terminal domain.Likewise, where the enhanced targeting domain is in the amino-terminalposition, an amino acid sequence comprising a start methionine and aprotease cleavage site may be operably-linked in situations in which theenhanced targeting domain requires a free amino terminus, see, e.g.,Shengwen Li et al., Degradable Clostridial Toxins, International PatentApplication Publication WO 2006/026780 (Mar. 9, 2006). In addition, itis known in the art that when adding a polypeptide that isoperably-linked to the amino terminus of another polypeptide comprisingthe start methionine that the original methionine residue can bedeleted.

Thus, in an embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising aClostridial toxin enzymatic domain, a Clostridial toxin translocationdomain, a Clostridial toxin translocation facilitating domain and anenhanced targeting domain. In an aspect of this embodiment, a modifiedClostridial toxin can comprise an amino to carboxyl single polypeptidelinear order comprising a Clostridial toxin enzymatic domain, a proteasecleavage site, a Clostridial toxin translocation domain, a Clostridialtoxin translocation facilitating domain and an enhanced targetingdomain. In another aspect of this embodiment, a modified Clostridialtoxin can comprise an amino to carboxyl single polypeptide linear ordercomprising a Clostridial toxin enzymatic domain, an endogenous proteasecleavage site, a Clostridial toxin translocation domain, a Clostridialtoxin translocation facilitating domain and an enhanced targetingdomain. In another aspect of this embodiment, a modified Clostridialtoxin can comprise an amino to carboxyl single polypeptide linear ordercomprising a Clostridial toxin enzymatic domain, an exogenous proteasecleavage site, a Clostridial toxin translocation domain, a Clostridialtoxin translocation facilitating domain and an enhanced targetingdomain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising aClostridial toxin enzymatic domain, an enhanced targeting domain, aClostridial toxin translocation domain and a Clostridial toxintranslocation facilitating domain. In an aspect of this embodiment, amodified Clostridial toxin can comprise an amino to carboxyl singlepolypeptide linear order comprising a Clostridial toxin enzymaticdomain, a protease cleavage site, an enhanced targeting domain, aClostridial toxin translocation domain and a Clostridial toxintranslocation facilitating domain. In another aspect of this embodiment,a modified Clostridial toxin can comprise an amino to carboxyl singlepolypeptide linear order comprising a Clostridial toxin enzymaticdomain, an endogenous protease cleavage site, an enhanced targetingdomain, a Clostridial toxin translocation domain and a Clostridial toxintranslocation facilitating domain. In another aspect of this embodiment,a modified Clostridial toxin can comprise an amino to carboxyl singlepolypeptide linear order comprising a Clostridial toxin enzymaticdomain, an exogenous protease cleavage site, an enhanced targetingdomain, a Clostridial toxin translocation domain and a Clostridial toxintranslocation facilitating domain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising an enhancedtargeting domain, a Clostridial toxin translocation domain, aClostridial toxin translocation facilitating domain and a Clostridialtoxin enzymatic domain. In an aspect of this embodiment, a modifiedClostridial toxin can comprise an amino to carboxyl single polypeptidelinear order comprising an enhanced targeting domain, a Clostridialtoxin translocation domain, a Clostridial toxin translocationfacilitating domain, a protease cleavage site and a Clostridial toxinenzymatic domain. In another aspect of this embodiment, a modifiedClostridial toxin can comprise an amino to carboxyl single polypeptidelinear order comprising an enhanced targeting domain, a Clostridialtoxin translocation domain, a Clostridial toxin translocationfacilitating domain, an endogenous protease cleavage site and aClostridial toxin enzymatic domain. In another aspect of thisembodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising an enhancedtargeting domain, a Clostridial toxin translocation domain, aClostridial toxin translocation facilitating domain, an exogenousprotease cleavage site and a Clostridial toxin enzymatic domain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising an enhancedtargeting domain, a Clostridial toxin enzymatic domain, a Clostridialtoxin translocation domain and a Clostridial toxin translocationfacilitating domain. In an aspect of this embodiment, a modifiedClostridial toxin can comprise an amino to carboxyl single polypeptidelinear order comprising an enhanced targeting domain, a Clostridialtoxin enzymatic domain, a protease cleavage site, a Clostridial toxintranslocation domain and a Clostridial toxin translocation facilitatingdomain. In another aspect of this embodiment, a modified Clostridialtoxin can comprise an amino to carboxyl single polypeptide linear ordercomprising an enhanced targeting domain, a Clostridial toxin enzymaticdomain, an endogenous protease cleavage site, a Clostridial toxintranslocation domain and a Clostridial toxin translocation facilitatingdomain. In another aspect of this embodiment, a modified Clostridialtoxin can comprise an amino to carboxyl single polypeptide linear ordercomprising an enhanced targeting domain, a Clostridial toxin enzymaticdomain, an exogenous protease cleavage site, a Clostridial toxintranslocation domain and a Clostridial toxin translocation facilitatingdomain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising aClostridial toxin translocation domain, a Clostridial toxintranslocation facilitating domain, a Clostridial toxin enzymatic domainand an enhanced targeting domain. In an aspect of this embodiment, amodified Clostridial toxin can comprise an amino to carboxyl singlepolypeptide linear order comprising a Clostridial toxin translocationdomain, a Clostridial toxin translocation facilitating domain, aprotease cleavage site, a Clostridial toxin enzymatic domain and anenhanced targeting domain. In another aspect of this embodiment, amodified Clostridial toxin can comprise an amino to carboxyl singlepolypeptide linear order comprising a Clostridial toxin translocationdomain, a Clostridial toxin translocation facilitating domain, anendogenous protease cleavage site, a Clostridial toxin enzymatic domainand an enhanced targeting domain. In another aspect of this embodiment,a modified Clostridial toxin can comprise an amino to carboxyl singlepolypeptide linear order comprising a Clostridial toxin translocationdomain, a Clostridial toxin translocation facilitating domain, anexogenous protease cleavage site, a Clostridial toxin enzymatic domainand an enhanced targeting domain.

Aspects of the present invention provide, in part modified Clostridialtoxins. Non-limiting examples of Clostridial toxin modificationsdisclosed in the present specification include, e.g., addition of anenhanced targeting domain, addition of a protease cleavage site,rearrangement of the enzymatic, translocation and binding domains andaddition of a spacer region. It is understood that all suchmodifications do not substantially affect the ability of a modifiedClostridial toxin to intoxicate a cell. As used herein, the term “do notsubstantially affect” means a modified Clostridial toxin can stillexecute the overall cellular mechanism whereby a Clostridial toxinenters a neuron and inhibits neurotransmitter release and encompassesthe binding of a Clostridial toxin to a low or high affinity receptorcomplex, the internalization of the toxin/receptor complex, thetranslocation of the Clostridial toxin light chain into the cytoplasmand the enzymatic modification of a Clostridial toxin substrate. Inaspects of this embodiment, the modified Clostridial toxin is, e.g., atleast 10% as toxic as a naturally-occurring Clostridial toxin, at least20% as toxic as a naturally-occurring Clostridial toxin, at least 30% astoxic as a naturally-occurring Clostridial toxin, at least 40% as toxicas a naturally-occurring Clostridial toxin, at least 50% as toxic as anaturally-occurring Clostridial toxin, at least 60% as toxic as anaturally-occurring Clostridial toxin, at least 70% as toxic as anaturally-occurring Clostridial toxin, at least 80% as toxic as anaturally-occurring Clostridial toxin, at least 90% as toxic as anaturally-occurring Clostridial toxin or at least 95% as toxic as anaturally-occurring Clostridial toxin. In aspects of this embodiment,the modified Clostridial toxin is, e.g., at most 10% as toxic as anaturally-occurring Clostridial toxin, at most 20% as toxic as anaturally-occurring Clostridial toxin, at most 30% as toxic as anaturally-occurring Clostridial toxin, at most 40% as toxic as anaturally-occurring Clostridial toxin, at most 50% as toxic as anaturally-occurring Clostridial toxin, at most 60% as toxic as anaturally-occurring Clostridial toxin, at most 70% as toxic as anaturally-occurring Clostridial toxin, at most 80% as toxic as anaturally-occurring Clostridial toxin, at most 90% as toxic as anaturally-occurring Clostridial toxin or at most 95% as toxic as anaturally-occurring Clostridial toxin.

Another aspect of the present invention provides polynucleotidemolecules encoding modified Clostridial toxins disclosed in the presentspecification. It is envisioned that any and all modified Clostridialtoxin disclosed in the present specification can be encoded by apolynucleotide molecule.

Aspects of the present invention provide, in part polynucleotidemolecules. As used herein, the term “polynucleotide molecule” issynonymous with “nucleic acid molecule” and means a polymeric form ofnucleotides, such as, e.g., ribonucleotides and deoxyribonucleotides, ofany length. It is envisioned that any and all polynucleotide moleculesthat can encode a modified Clostridial toxin disclosed in the presentspecification can be useful, including, without limitationnaturally-occurring and non-naturally-occurring DNA molecules andnaturally-occurring and non-naturally-occurring RNA molecules.Non-limiting examples of naturally-occurring and non-naturally-occurringDNA molecules include single-stranded DNA molecules, double-stranded DNAmolecules, genomic DNA molecules, cDNA molecules, vector constructs,such as, e.g., plasmid constructs, phagmid constructs, bacteriophageconstructs, retroviral constructs and artificial chromosome constructs.Non-limiting examples of naturally-occurring and non-naturally-occurringRNA molecules include single-stranded RNA, double stranded RNA and mRNA.

Thus, in an embodiment, a polynucleotide molecule encodes a modifiedClostridial toxin disclosed in the present specification.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising a Clostridial toxin enzymaticdomain disclosed in the present specification. In an aspect of thisembodiment, a polynucleotide molecule encoding a modified Clostridialtoxin enzymatic domain comprises a naturally occurring Clostridial toxinenzymatic domain variant, such as, e.g., a Clostridial toxin enzymaticdomain isoform or a Clostridial toxin enzymatic domain subtype. Inanother aspect of this embodiment, a polynucleotide molecule encoding aClostridial toxin enzymatic domain comprises a non-naturally occurringClostridial toxin enzymatic domain variant, such as, e.g., aconservative Clostridial toxin enzymatic domain variant, anon-conservative Clostridial toxin enzymatic domain variant or an activeClostridial toxin enzymatic domain fragment, or any combination thereof.In other aspects of this embodiment, a polynucleotide molecule encodinga Clostridial toxin enzymatic domain comprises a BoNT/A enzymaticdomain, a BoNT/B enzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/Denzymatic domain, a BoNT/E enzymatic domain, a BoNT/F enzymatic domain,a BoNT/G enzymatic domain, a TeNT enzymatic domain, or active fragmentthereof.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising a Clostridial toxin translocationdomain disclosed in the present specification. In an aspect of thisembodiment, a polynucleotide molecule encoding a modified Clostridialtoxin translocation domain comprises a naturally occurring Clostridialtoxin translocation domain variant, such as, e.g., a Clostridial toxintranslocation domain isoform or a Clostridial toxin translocation domainsubtype. In another aspect of this embodiment, a polynucleotide moleculeencoding a Clostridial toxin translocation domain comprises anon-naturally occurring Clostridial toxin translocation domain variant,such as, e.g., a conservative Clostridial toxin translocation domainvariant, a non-conservative Clostridial toxin translocation domainvariant or an active Clostridial toxin translocation domain fragment, orany combination thereof. In other aspects of this embodiment, apolynucleotide molecule encoding a Clostridial toxin translocationdomain comprises a BoNT/A translocation domain, a BoNT/B translocationdomain, a BoNT/C1 translocation domain, a BoNT/D translocation domain, aBoNT/E translocation domain, a BoNT/F translocation domain, a BoNT/Gtranslocation domain, a TeNT translocation domain, or active fragmentthereof.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising a translocation facilitatingdomain disclosed in the present specification. In an aspect of thisembodiment, a polynucleotide molecule encoding a translocationfacilitating domain comprises a naturally occurring Clostridial toxintranslocation facilitating domain variant, such as, e.g., a Clostridialtoxin translocation facilitating domain isoform or a Clostridial toxintranslocation facilitating domain subtype. In another aspect of thisembodiment, a polynucleotide molecule encoding a translocationfacilitating domain comprises a non-naturally occurring Clostridialtoxin translocation facilitating domain variant, such as, e.g., aconservative Clostridial toxin translocation facilitating domainvariant, a non-conservative Clostridial toxin translocation facilitatingdomain variant or an active Clostridial toxin translocation facilitatingdomain fragment, or any combination thereof. In other aspects of thisembodiment, a polynucleotide molecule encoding a Clostridial toxintranslocation facilitating domain comprises a BoNT/A translocationfacilitating domain, a BoNT/B translocation facilitating domain, aBoNT/C1 translocation facilitating domain, a BoNT/D translocationfacilitating domain, a BoNT/E translocation facilitating domain, aBoNT/F translocation facilitating domain, a BoNT/G translocationfacilitating domain, a TeNT translocation facilitating domain, or activefragment thereof. In yet another aspect of this embodiment, apolynucleotide molecule encoding a translocation domain comprises anaturally occurring enveloped virus fusogenic peptide domain variant,such as, e.g., an enveloped virus fusogenic peptide domain isoform or anenveloped virus fusogenic peptide domain subtype. In another aspect ofthis embodiment, a polynucleotide molecule encoding a translocationdomain comprises a non-naturally occurring enveloped virus fusogenicpeptide domain variant, such as, e.g., a conservative enveloped virusfusogenic peptide domain variant, a non-conservative enveloped virusfusogenic peptide domain variant or an active enveloped virus fusogenicpeptide domain fragment, or any combination thereof. In other aspects ofthis embodiment, a polynucleotide molecule encoding an enveloped virusfusogenic peptide domain comprisesan influenzavirus fusogenic peptidedomain, an alphavirus fusogenic peptide domain, a vesiculovirusfusogenic peptide domain, a respirovirus fusogenic peptide domain, amorbillivirus fusogenic peptide domain, an avulavirus fusogenic peptidedomain, a henipavirus fusogenic peptide domain, a metapneumovirusfusogenic peptide domain, a foamy virus fusogenic peptide domain, oractive fragment thereof.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising an enhanced targeting domaindisclosed in the present specification. In an aspect of this embodiment,a polynucleotide molecule encoding an enhanced targeting domaincomprises a modified Clostridial toxin targeting domain with enhancedbinding activity disclosed in the present specification. In an aspect ofthis embodiment, a polynucleotide molecule encoding a modifiedClostridial toxin targeting domain with enhanced binding activitycomprises a non-naturally occurring Clostridial toxin targeting domainvariant with enhanced binding activity, such as, e.g., a conservativeClostridial toxin targeting domain variant with enhanced bindingactivity, a non-conservative Clostridial toxin targeting domain variantwith enhanced binding activity or an active Clostridial toxin targetingdomain fragment with enhanced binding activity, or any combinationthereof. In other aspects of this embodiment, a polynucleotide moleculeencoding a Clostridial toxin targeting domain with enhanced bindingactivity comprises a BoNT/A targeting domain with enhanced bindingactivity, a BoNT/B targeting domain with enhanced binding activity, aBoNT/C1 targeting domain with enhanced binding activity, a BoNT/Dtargeting domain with enhanced binding activity, a BoNT/E targetingdomain with enhanced binding activity, a BoNT/F targeting domain withenhanced binding activity, a BoNT/G targeting domain with enhancedbinding activity, a TeNT targeting domain with enhanced bindingactivity, or active fragment thereof.

In an aspect of this embodiment, an enhanced targeting domain comprisesa Clostridial NAP disclosed in the present specification. In an aspectof this embodiment, a polynucleotide molecule encoding a Clostridial NAPcomprises a naturally occurring Clostridial NAP variant, such as, e.g.,a Clostridial NAP isoform or a Clostridial NAP subtype. In an aspect ofthis embodiment, a polynucleotide molecule encoding a NAP comprises anon-naturally occurring Clostridial NAP variant, such as, e.g., aconservative Clostridial NAP variant, a non-conservative Clostridial NAPvariant or an active Clostridial NAP fragment, or any combinationthereof. In other aspects of this embodiment, a polynucleotide moleculeencoding a Clostridial NAP comprises a Clostridial botulinum serotype AHA-33, a Clostridial botulinum serotype B HA-33, a Clostridial botulinumserotype C1 HA-33, a Clostridial botulinum serotype D HA-33, aClostridial botulinum serotype A HA-17, a Clostridial botulinum serotypeB HA-17, a Clostridial botulinum serotype C1 HA-17, a Clostridialbotulinum serotype D HA-17, a Clostridial botulinum serotype A NTNH, aClostridial botulinum serotype B NTNH, a Clostridial botulinum serotypeC1 NTNH, a Clostridial botulinum serotype D NTNH, a Clostridialbotulinum serotype E NTNH, a Clostridial botulinum serotype F NTNH and aClostridial botulinum serotype G NTNH.

In an aspect of this embodiment, an enhanced targeting domain comprisesa Clostridial NAP with enhanced binding activity disclosed in thepresent specification. In an aspect of this embodiment, a polynucleotidemolecule encoding a NAP with enhanced binding activity comprises anon-naturally occurring Clostridial NAP variant with enhanced bindingactivity, such as, e.g., a conservative Clostridial NAP variant withenhanced binding activity, a non-conservative Clostridial NAP variantwith enhanced binding activity or an active Clostridial NAP fragmentwith enhanced binding activity, or any combination thereof. In otheraspects of this embodiment, a polynucleotide molecule encoding aClostridial NAP comprises a Clostridial botulinum serotype A HA-33 withenhanced binding activity, a Clostridial botulinum serotype B HA-33 withenhanced binding activity, a Clostridial botulinum serotype C1 HA-33with enhanced binding activity, a Clostridial botulinum serotype D HA-33with enhanced binding activity, a Clostridial botulinum serotype A HA-17with enhanced binding activity, a Clostridial botulinum serotype B HA-17with enhanced binding activity, a Clostridial botulinum serotype C1HA-17 with enhanced binding activity, a Clostridial botulinum serotype DHA-17 with enhanced binding activity, a Clostridial botulinum serotype ANTNH with enhanced binding activity, a Clostridial botulinum serotype BNTNH with enhanced binding activity, a Clostridial botulinum serotype C1NTNH with enhanced binding activity, a Clostridial botulinum serotype DNTNH with enhanced binding activity, a Clostridial botulinum serotype ENTNH with enhanced binding activity, a Clostridial botulinum serotype FNTNH with enhanced binding activity and a Clostridial botulinum serotypeG NTNH with enhanced binding activity.

In an aspect of this embodiment, an enhanced targeting domain comprisesa FGF disclosed in the present specification. In an aspect of thisembodiment, a polynucleotide molecule encoding a FGF comprises anaturally occurring FGF variant, such as, e.g., a FGF isoform or a FGFsubtype. In an aspect of this embodiment, a polynucleotide moleculeencoding a FGF comprises a non-naturally occurring FGF variant, such as,e.g., a conservative FGF variant, a non-conservative FGF variant or anactive FGF fragment, or any combination thereof. In other aspects ofthis embodiment, a polynucleotide molecule encoding a FGF comprises aFGF-1, a FGF-2, a FGF-4, a FGF-8, a FGF-9, a FGF-17 and a FGF-18.

In an aspect of this embodiment, an enhanced targeting domain comprisesa FGF with enhanced binding activity disclosed in the presentspecification. In an aspect of this embodiment, a polynucleotidemolecule encoding a FGF with enhanced binding activity comprises anon-naturally occurring FGF variant with enhanced binding activity, suchas, e.g., a conservative FGF variant with enhanced binding activity, anon-conservative FGF variant with enhanced binding activity or an activeFGF fragment with enhanced binding activity, or any combination thereof.In other aspects of this embodiment, a polynucleotide molecule encodinga FGF with enhanced binding activity comprises a FGF-1 with enhancedbinding activity, a FGF-2 with enhanced binding activity, a FGF-4 withenhanced binding activity, a FGF-8 with enhanced binding activity, aFGF-9 with enhanced binding activity, a FGF-17 with enhanced bindingactivity and a FGF-18 with enhanced binding activity.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising a protease cleavage site disclosedin the present specification. In an aspect of this embodiment, apolynucleotide molecule encoding a protease cleavage site comprises anendogenous Clostridial toxin protease site. In aspects of thisembodiment, a polynucleotide molecule encoding an endogenous Clostridialtoxin protease site can be, e.g., a BoNT/A di-chain loop proteasecleavage site, a BoNT/B di-chain loop protease cleavage site, a BoNT/C1di-chain loop protease cleavage site, a BoNT/D di-chain loop proteasecleavage site, a BoNT/E di-chain loop protease cleavage site, a BoNT/Fdi-chain loop protease cleavage site, a BoNT/G di-chain loop proteasecleavage site or a TeNT di-chain loop protease cleavage site. In anotheraspect of this embodiment, a polynucleotide molecule encoding a proteasecleavage site comprises an exogenous Clostridial toxin protease site. Inaspects of this embodiment, a polynucleotide molecule encoding anexogenous Clostridial toxin protease site can be, e.g., a bovineenterokinase protease cleavage site, a Tobacco Etch Virus proteasecleavage site, a Human Rhinovirus 3C protease cleavage site, aSUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site, aCoagulation Factor Xa protease cleavage site or a Clostridial toxinsubstrate cleavage site, such as, e.g., a BoNT/A substrate cleavagesite, a BoNT/B substrate cleavage site, a BoNT/C1 substrate cleavagesite, a BoNT/D substrate cleavage site, a BoNT/E substrate cleavagesite, a BoNT/F substrate cleavage site, a BoNT/G substrate cleavage siteor a TeNT substrate cleavage site.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising a flexible spacer disclosed in thepresent specification. In an aspect of this embodiment, a polynucleotidemolecule encoding a flexible spacer can comprise a G-spacer, a A-spaceror any combination thereof.

In another embodiment, a polynucleotide molecule encodes, in part, amodified Clostridial toxin comprising an epitope-binding regiondisclosed in the present specification. In an aspect of this embodiment,a polynucleotide molecule encoding an epitope-binding region cancomprise a FLAG, Express™, a human Influenza virus hemagluttinin (HA), ahuman p62^(c-Myc) protein (c-MYC), a Vesicular Stomatitis VirusGlycoprotein (VSV-G), a Substance P, a glycoprotein-D precursor ofHerpes simplex virus (HSV), a V5, a AU1, a AU5 (SEQ ID NO: 85), apolyhistidine (HIS), or any combination thereof.

Well-established molecular biology techniques that may be necessary tomake a polynucleotide molecule encoding a modified Clostridial toxindisclosed in the present specification including, but not limited to,procedures involving polymerase chain reaction (PCR) amplification,restriction enzyme reactions, agarose gel electrophoresis, nucleic acidligation, bacterial transformation, nucleic acid purification, nucleicacid sequencing and recombination-based techniques are routineprocedures well within the scope of one skilled in the art and from theteaching herein. Non-limiting examples of specific protocols necessaryto make a polynucleotide molecule encoding a modified Clostridial toxinare described in e.g., MOLECULAR CLONING A LABORATORY MANUAL, supra,(2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubelet al., eds. John Wiley & Sons, 2004). Additionally, a variety ofcommercially available products useful for making a polynucleotidemolecule encoding a modified Clostridial toxin are widely available.These protocols are routine procedures well within the scope of oneskilled in the art and from the teaching herein.

Another aspect of the present invention provides a method of producing amodified Clostridial toxin disclosed in the present specification, suchmethod comprising the step of expressing a polynucleotide moleculeencoding a modified Clostridial toxin in a cell. Another aspect of thepresent invention provides a method of producing a modified Clostridialtoxin disclosed in the present specification, such method comprising thesteps of introducing an expression construct comprising a polynucleotidemolecule encoding a modified Clostridial toxin into a cell andexpressing the expression construct in the cell.

The methods disclosed in the present specification include, in part, amodified Clostridial toxin. It is envisioned that any and all modifiedClostridial toxins disclosed in the present specification can beproduced using the methods disclosed in the present specification. It isalso envisioned that any and all polynucleotide molecules encoding amodified Clostridial toxins disclosed in the present specification canbe useful in producing a modified Clostridial toxins disclosed in thepresent specification using the methods disclosed in the presentspecification.

The methods disclosed in the present specification include, in part, anexpression construct. An expression construct comprises a polynucleotidemolecule disclosed in the present specification operably-linked to anexpression vector useful for expressing the polynucleotide molecule in acell or cell-free extract. A wide variety of expression vectors can beemployed for expressing a polynucleotide molecule encoding a modifiedClostridial toxin, including, without limitation, a viral expressionvector; a prokaryotic expression vector; eukaryotic expression vectors,such as, e.g., a yeast expression vector, an insect expression vectorand a mammalian expression vector; and a cell-free extract expressionvector. It is further understood that expression vectors useful topractice aspects of these methods may include those which express amodified Clostridial toxin under control of a constitutive,tissue-specific, cell-specific or inducible promoter element, enhancerelement or both. Non-limiting examples of expression vectors, along withwell-established reagents and conditions for making and using anexpression construct from such expression vectors are readily availablefrom commercial vendors that include, without limitation, BDBiosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, SanDiego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMDBiosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; andStratagene, La Jolla, Calif. The selection, making and use of anappropriate expression vector are routine procedures well within thescope of one skilled in the art and from the teachings herein.

Thus, aspects of this embodiment include, without limitation, a viralexpression vector operably-linked to a polynucleotide molecule encodinga modified Clostridial toxin; a prokaryotic expression vectoroperably-linked to a polynucleotide molecule encoding a modifiedClostridial toxin; a yeast expression vector operably-linked to apolynucleotide molecule encoding a modified Clostridial toxin; an insectexpression vector operably-linked to a polynucleotide molecule encodinga modified Clostridial toxin; and a mammalian expression vectoroperably-linked to a polynucleotide molecule encoding a modifiedClostridial toxin. Other aspects of this embodiment include, withoutlimitation, expression constructs suitable for expressing a modifiedClostridial toxin disclosed in the present specification using acell-free extract comprising a cell-free extract expression vectoroperably linked to a polynucleotide molecule encoding a modifiedClostridial toxin. Other aspects of this embodiment include, withoutlimitation, expression constructs comprising polynucleotide moleculescomprising any one of SEQ ID NO: 109 through SEQ ID NO: 132 and SEQ IDNO: 136 through SEQ ID NO: 159. Other aspects of this embodimentinclude, without limitation, expression constructs comprisingpolynucleotide molecules encoding a modified Clostridial toxincomprising any one of SEQ ID NO: 85 through SEQ ID NO: 108.

The methods disclosed in the present specification include, in part, acell. It is envisioned that any and all cells can be used. Thus, aspectsof this embodiment include, without limitation, prokaryotic cellsincluding, without limitation, strains of aerobic, microaerophilic,capnophilic, facultative, anaerobic, gram-negative and gram-positivebacterial cells such as those derived from, e.g., Escherichia coli,Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis,Clostridia perfringens, Clostridia difficile, Caulobacter crescentus,Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls,Neisseria meningitidis, Pseudomonas fluorescens and Salmonellatyphimurium; and eukaryotic cells including, without limitation, yeaststrains, such as, e.g., those derived from Pichia pastoris, Pichiamethanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomycescerevisiae and Yarrowia lipolytica; insect cells and cell lines derivedfrom insects, such as, e.g., those derived from Spodoptera frugiperda,Trichoplusia ni, Drosophila melanogaster and Manduca sexta; andmammalian cells and cell lines derived from mammalian cells, such as,e.g., those derived from mouse, rat, hamster, porcine, bovine, equine,primate and human. Cell lines may be obtained from the American TypeCulture Collection, European Collection of Cell Cultures and the GermanCollection of Microorganisms and Cell Cultures. Non-limiting examples ofspecific protocols for selecting, making and using an appropriate cellline are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F.A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES:FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer AcademicPublishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUESOF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUECULTURE: LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley andSons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASICTECHNIQUE (Wiley-Liss, 4^(th) ed. 2000); ANIMAL CELL CULTURE: APRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press,3^(rd) ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001);BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press,2^(nd) ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra,(2004). These protocols are routine procedures within the scope of oneskilled in the art and from the teaching herein.

The methods disclosed in the present specification include, in part,introducing into a cell a polynucleotide molecule. A polynucleotidemolecule introduced into a cell can be transiently or stably maintainedby that cell. Stably-maintained polynucleotide molecules may beextra-chromosomal and replicate autonomously, or they may be integratedinto the chromosomal material of the cell and replicatenon-autonomously. It is envisioned that any and all methods forintroducing a polynucleotide molecule disclosed in the presentspecification into a cell can be used. Methods useful for introducing anucleic acid molecule into a cell include, without limitation,chemical-mediated transfection such as, e.g., calciumphosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated,lipid-mediated, polyethyleneimine (PEI)-mediated, polylysine-mediatedand polybrene-mediated; physical-mediated transfection, such as, e.g.,biolistic particle delivery, microinjection, protoplast fusion andelectroporation; and viral-mediated transfection, such as, e.g.,retroviral-mediated transfection, see, e.g., Introducing Cloned Genesinto Cultured Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds.,Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed. 2001). Oneskilled in the art understands that selection of a specific method tointroduce an expression construct into a cell will depend, in part, onwhether the cell will transiently contain an expression construct orwhether the cell will stably contain an expression construct. Theseprotocols are routine procedures within the scope of one skilled in theart and from the teaching herein.

In an aspect of this embodiment, a chemical-mediated method, termedtransfection, is used to introduce a polynucleotide molecule encoding amodified Clostridial toxin into a cell. In chemical-mediated methods oftransfection the chemical reagent forms a complex with the nucleic acidthat facilitates its uptake into the cells. Such chemical reagentsinclude, without limitation, calcium phosphate-mediated, see, e.g.,Martin Jordan & Florian Worm, Transfection of adherent and suspendedcells by calcium phosphate, 33(2) Methods 136-143 (2004);diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, cationicpolymer-mediated like polyethyleneimine (PEI)-mediated andpolylysine-mediated and polybrene-mediated, see, e.g., Chun Zhang etal., Polyethylenimine strategies for plasmid delivery to brain-derivedcells, 33(2) Methods 144-150 (2004). Such chemical-mediated deliverysystems can be prepared by standard methods and are commerciallyavailable, see, e.g., CellPhect Transfection Kit (Amersham Biosciences,Piscataway, N.J.); Mammalian Transfection Kit, Calcium phosphate andDEAE Dextran, (Stratagene, Inc., La Jolla, Calif.); Lipofectamine™Transfection Reagent (Invitrogen, Inc., Carlsbad, Calif.); ExGen 500Transfection kit (Fermentas, Inc., Hanover, Md.), and SuperFect andEffectene Transfection Kits (Qiagen, Inc., Valencia, Calif.).

In another aspect of this embodiment, a physical-mediated method is usedto introduce a polynucleotide molecule encoding a modified Clostridialtoxin into a cell. Physical techniques include, without limitation,electroporation, biolistic and microinjection. Biolistics andmicroinjection techniques perforate the cell wall in order to introducethe nucleic acid molecule into the cell, see, e.g., Jeike E. Biewenga etal., Plasmid-mediated gene transfer in neurons using the biolisticstechnique, 71(1) J. Neurosci. Methods. 67-75 (1997); and John O'Brien &Sarah C. R. Lummis, Biolistic and diolistic transfection: using the genegun to deliver DNA and lipophilic dyes into mammalian cells, 33(2)Methods 121-125 (2004). Electroporation, also termedelectropermeabilization, uses brief, high-voltage, electrical pulses tocreate transient pores in the membrane through which the nucleic acidmolecules enter and can be used effectively for stable and transienttransfections of all cell types, see, e.g., M. Golzio et al., In vitroand in vivo electric field-mediated permeabilization, gene transfer, andexpression, 33(2) Methods 126-135 (2004); and Oliver Gresch et al., Newnon-viral method for gene transfer into primary cells, 33(2) Methods151-163 (2004).

In another aspect of this embodiment, a viral-mediated method, termedtransduction, is used to introduce a polynucleotide molecule encoding amodified Clostridial toxin into a cell. In viral-mediated methods oftransient transduction, the process by which viral particles infect andreplicate in a host cell has been manipulated in order to use thismechanism to introduce a nucleic acid molecule into the cell.Viral-mediated methods have been developed from a wide variety ofviruses including, without limitation, retroviruses, adenoviruses,adeno-associated viruses, herpes simplex viruses, picornaviruses,alphaviruses and baculoviruses, see, e.g., Armin Blesch, Lentiviral andMLV based retroviral vectors for ex vivo and in vivo gene transfer,33(2) Methods 164-172 (2004); and Maurizio Federico, From lentivirusesto lentivirus vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M.Poeschla, Non-primate lentiviral vectors, 5(5) Curr. Opin. Mol. Ther.529-540 (2003); Karim Benihoud et al, Adenovirus vectors for genedelivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H. Bueler,Adeno-associated viral vectors for gene transfer and gene therapy,380(6) Biol. Chem. 613-622 (1999); Chooi M. Lai et al., Adenovirus andadeno-associated virus vectors, 21(12) DNA Cell Biol. 895-913 (2002);Edward A. Burton et al., Gene delivery using herpes simplex virusvectors, 21(12) DNA Cell Biol. 915-936 (2002); Paola Grandi et al.,Targeting HSV amplicon vectors, 33(2) Methods 179-186 (2004); IlyaFrolov et al., Alphavirus-based expression vectors: strategies andapplications, 93(21) Proc. Natl. Acad. Sci. U.S.A. 11371-11377 (1996);Markus U. Ehrengruber, Alphaviral gene transfer in neurobiology, 59(1)Brain Res. Bull. 13-22 (2002); Thomas A. Kost & J. Patrick Condreay,Recombinant baculoviruses as mammalian cell gene-delivery vectors, 20(4)Trends Biotechnol. 173-180 (2002); and A. Huser & C. Hofmann,Baculovirus vectors: novel mammalian cell gene-delivery vehicles andtheir applications, 3(1) Am. J. Pharmacogenomics 53-63 (2003).

Adenoviruses, which are non-enveloped, double-stranded DNA viruses, areoften selected for mammalian cell transduction because adenoviruseshandle relatively large polynucleotide molecules of about 36 kb, areproduced at high titer, and can efficiently infect a wide variety ofboth dividing and non-dividing cells, see, e.g., Wim T. J. M. C. Hermenset al., Transient gene transfer to neurons and glia: analysis ofadenoviral vector performance in the CNS and PNS, 71(1) J. Neurosci.Methods 85-98 (1997); and Hiroyuki Mizuguchi et al., Approaches forgenerating recombinant adenovirus vectors, 52(3) Adv. Drug Deliv. Rev.165-176 (2001). Transduction using adenoviral-based system do notsupport prolonged protein expression because the nucleic acid moleculeis carried from an episome in the cell nucleus, rather than beingintegrated into the host cell chromosome. Adenoviral vector systems andspecific protocols for how to use such vectors are disclosed in, e.g.,ViraPower™ Adenoviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and ViraPower™ Adenoviral Expression System Instruction Manual25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and AdEasy™Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif.) andAdEasy™ Adenoviral Vector System Instruction Manual 064004f, Stratagene,Inc.

Nucleic acid molecule delivery can also use single-stranded RNAretroviruses, such as, e.g., oncoretroviruses and lentiviruses.Retroviral-mediated transduction often produce transduction efficienciesclose to 100%, can easily control the proviral copy number by varyingthe multiplicity of infection (MOI), and can be used to eithertransiently or stably transduce cells, see, e.g., Tiziana Tonini et al.,Transient production of retroviral- and lentiviral-based vectors for thetransduction of Mammalian cells, 285 Methods Mol. Biol. 141-148 (2004);Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivoand in vivo gene transfer, 33(2) Methods 164-172 (2004); FélixRecillas-Targa, Gene transfer and expression in mammalian cell lines andtransgenic animals, 267 Methods Mol. Biol. 417-433 (2004); and RolandWolkowicz et al., Lentiviral vectors for the delivery of DNA intomammalian cells, 246 Methods Mol. Biol. 391-411 (2004). Retroviralparticles consist of an RNA genome packaged in a protein capsid,surrounded by a lipid envelope. The retrovirus infects a host cell byinjecting its RNA into the cytoplasm along with the reversetranscriptase enzyme. The RNA template is then reverse transcribed intoa linear, double stranded cDNA that replicates itself by integratinginto the host cell genome. Viral particles are spread both vertically(from parent cell to daughter cells via the provirus) as well ashorizontally (from cell to cell via virions). This replication strategyenables long-term persistent expression since the nucleic acid moleculesof interest are stably integrated into a chromosome of the host cell,thereby enabling long-term expression of the protein. For instance,animal studies have shown that lentiviral vectors injected into avariety of tissues produced sustained protein expression for more than 1year, see, e.g., Luigi Naldini et al., In vivo gene delivery and stabletransduction of non-dividing cells by a lentiviral vector, 272(5259)Science 263-267 (1996). The Oncoretroviruses-derived vector systems,such as, e.g., Moloney murine leukemia virus (MoMLV), are widely usedand infect many different non-dividing cells. Lentiviruses can alsoinfect many different cell types, including dividing and non-dividingcells and possess complex envelope proteins, which allows for highlyspecific cellular targeting.

Retroviral vectors and specific protocols for how to use such vectorsare disclosed in, e.g., U.S. Pat. Nos. ______ Manfred Gossen & HermannBujard, Tight control of gene expression in eukaryotic cells bytetracycline-responsive promoters, U.S. Pat. No. 5,464,758 (Nov. 7,1995) and Hermann Bujard & Manfred Gossen, Methods for regulating geneexpression, U.S. Pat. No. 5,814,618 (Sep. 29, 1998) David S. Hogness,Polynucleotides encoding insect steroid hormone receptor polypeptidesand cells transformed with same, U.S. Pat. No. 5,514,578 (May 7, 1996)and David S. Hogness, Polynucleotide encoding insect ecdysone receptor,U.S. Pat. No. 6,245,531 (Jun. 12, 2001); Elisabetta Vegeto et al.,Progesterone receptor having C. terminal hormone binding domaintruncations, U.S. Pat. No. 5,364,791 (Nov. 15, 1994), Elisabetta Vegetoet al., Mutated steroid hormone receptors, methods for their use andmolecular switch for gene therapy, U.S. Pat. No. 5,874,534 (Feb. 23,1999) and Elisabetta Vegeto et al., Mutated steroid hormone receptors,methods for their use and molecular switch for gene therapy, U.S. Pat.No. 5,935,934 (Aug. 10, 1999). Furthermore, such viral delivery systemscan be prepared by standard methods and are commercially available, see,e.g., BD™ Tet-Off and Tet-On Gene Expression Systems (BDBiosciences-Clonetech, Palo Alto, Calif.) and BD™ Tet-Off and Tet-OnGene Expression Systems User Manual, PT3001-1, BD Biosciences Clonetech,(Mar. 14, 2003), GeneSwitch™ System (Invitrogen, Inc., Carlsbad, Calif.)and GeneSwitch™ System A Mifepristone-Regulated Expression System forMammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002);ViraPower™ Lentiviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and ViraPower™ Lentiviral Expression System Instruction Manual25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and CompleteControl® Retroviral Inducible Mammalian Expression System (Stratagene,La Jolla, Calif.) and Complete Control® Retroviral Inducible MammalianExpression System Instruction Manual, 064005e.

The methods disclosed in the present specification include, in part,expressing a modified Clostridial toxin from a polynucleotide molecule.It is envisioned that any of a variety of expression systems may beuseful for expressing a modified Clostridial toxin from a polynucleotidemolecule disclosed in the present specification, including, withoutlimitation, cell-based systems and cell-free expression systems.Cell-based systems include, without limitation, viral expressionsystems, prokaryotic expression systems, yeast expression systems,baculoviral expression systems, insect expression systems and mammalianexpression systems. Cell-free systems include, without limitation, wheatgerm extracts, rabbit reticulocyte extracts and E. coli extracts andgenerally are equivalent to the method disclosed herein. Expression of apolynucleotide molecule using an expression system can include any of avariety of characteristics including, without limitation, inducibleexpression, non-inducible expression, constitutive expression,viral-mediated expression, stably-integrated expression, and transientexpression. Expression systems that include well-characterized vectors,reagents, conditions and cells are well-established and are readilyavailable from commercial vendors that include, without limitation,Ambion, Inc. Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.;BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied Science,Indianapolis, Ind.; and Stratagene, La Jolla, Calif. Non-limitingexamples on the selection and use of appropriate heterologous expressionsystems are described in e.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH(S. J. Higgins and B. David Hames eds., Oxford University Press, 1999);Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS. USINGNATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and Meena Rai &Harish Padh, Expression Systems for Production of Heterologous Proteins,80(9) CURRENT SCIENCE 1121-1128, (2001). These protocols are routineprocedures well within the scope of one skilled in the art and from theteaching herein.

A variety of cell-based expression procedures are useful for expressinga modified Clostridial toxin encoded by polynucleotide moleculedisclosed in the present specification. Examples included, withoutlimitation, viral expression systems, prokaryotic expression systems,yeast expression systems, baculoviral expression systems, insectexpression systems and mammalian expression systems. Viral expressionsystems include, without limitation, the ViraPower™ Lentiviral(Invitrogen, Inc., Carlsbad, Calif.), the Adenoviral Expression Systems(Invitrogen, Inc., Carlsbad, Calif.), the AdEasy™ XL Adenoviral VectorSystem (Stratagene, La Jolla, Calif.) and the ViraPort® Retroviral GeneExpression System (Stratagene, La Jolla, Calif.). Non-limiting examplesof prokaryotic expression systems include the Champion™ pET ExpressionSystem (EMD Biosciences-Novagen, Madison, Wis.), the TriEx™ BacterialExpression System (EMD Biosciences-Novagen, Madison, Wis.), theQIAexpress® Expression System (QIAGEN, Inc.), and the Affinity® ProteinExpression and Purification System (Stratagene, La Jolla, Calif.). Yeastexpression systems include, without limitation, the EasySelect™ PichiaExpression Kit (Invitrogen, Inc., Carlsbad, Calif.), the YES-Echo™Expression Vector Kits (Invitrogen, Inc., Carlsbad, Calif.) and theSpECTRA™ S. pombe Expression System (Invitrogen, Inc., Carlsbad,Calif.). Non-limiting examples of baculoviral expression systems includethe BaculoDirect™ (Invitrogen, Inc., Carlsbad, Calif.), the Bac-to-Bac®(Invitrogen, Inc., Carlsbad, Calif.), and the BD BaculoGold™ (BDBiosciences-Pharmigen, San Diego, Calif.). Insect expression systemsinclude, without limitation, the Drosophila Expression System (DES®)(Invitrogen, Inc., Carlsbad, Calif.), InsectSelect™ System (Invitrogen,Inc., Carlsbad, Calif.) and InsectDirect™ System (EMDBiosciences-Novagen, Madison, Wis.). Non-limiting examples of mammalianexpression systems include the T-REx™ (Tetracycline-RegulatedExpression) System (Invitrogen, Inc., Carlsbad, Calif.), the Flp-In™T-REx™ System (Invitrogen, Inc., Carlsbad, Calif.), the pcDNA™ system(Invitrogen, Inc., Carlsbad, Calif.), the pSecTag2 system (Invitrogen,Inc., Carlsbad, Calif.), the Exchanger® System, InterPlay™ Mammalian TAPSystem (Stratagene, La Jolla, Calif.), Complete Control® InducibleMammalian Expression System (Stratagene, La Jolla, Calif.) andLacSwitch® II Inducible Mammalian Expression System (Stratagene, LaJolla, Calif.).

Another procedure of expressing a modified Clostridial toxin encoded bypolynucleotide molecule disclosed in the present specification employs acell-free expression system such as, without limitation, prokaryoticextracts and eukaryotic extracts. Non-limiting examples of prokaryoticcell extracts include the RTS 100 E. coli HY Kit (Roche Applied Science,Indianapolis, Ind.), the ActivePro In Vitro Translation Kit (Ambion,Inc., Austin, Tex.), the EcoPro™ System (EMD Biosciences-Novagen,Madison, Wis.) and the Expressway™ Plus Expression System (Invitrogen,Inc., Carlsbad, Calif.). Eukaryotic cell extract include, withoutlimitation, the RTS 100 Wheat Germ CECF Kit (Roche Applied Science,Indianapolis, Ind.), the TnT® Coupled Wheat Germ Extract Systems(Promega Corp., Madison, Wis.), the Wheat Germ IVT™ Kit (Ambion, Inc.,Austin, Tex.), the Retic Lysate IVT™ Kit (Ambion, Inc., Austin, Tex.),the PROTEINscript® II System (Ambion, Inc., Austin, Tex.) and the TnT®Coupled Reticulocyte Lysate Systems (Promega Corp., Madison, Wis.).

Aspects of the present invention can also be described as follows:

-   1. A modified Clostridial toxin comprising:    -   a) a Clostridial toxin enzymatic domain capable of executing an        enzymatic target modification step of a Clostridial toxin        intoxication process;    -   b) a Clostridial toxin translocation domain capable of executing        a translocation step of a Clostridial toxin intoxication        process;    -   c) a translocation facilitating domain capable of facilitating a        translocation step of a Clostridial toxin intoxication process;    -   d) an enhanced targeting domain comprising a modified        Clostridial H_(CC) targeting domain capable of executing a cell        binding step of a Clostridial toxin intoxication process; and    -   e) a protease cleavage site;    -   wherein cleavage of the protease cleavage site converts the        single-chain form of the modified Clostridial toxin into the        di-chain form; and    -   wherein the modified Clostridial H_(CC) targeting domain        exhibits enhanced binding activity for an endogenous Clostridial        toxin receptor relative to the binding activity of a        naturally-occurring Clostridial H_(CC) targeting domain from        which the modified Clostridial H_(CC) targeting domain is        derived.-   2. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin enzymatic domain,    the protease cleavage site, the Clostridial toxin translocation    domain, the translocation facilitating domain and the enhanced    targeting domain.-   3. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin enzymatic domain,    the protease cleavage site, the enhanced targeting domain, the    Clostridial toxin translocation domain and the translocation    facilitating domain.-   4. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the enhanced targeting domain, the    Clostridial toxin translocation domain, the translocation    facilitating domain, the protease cleavage site and the Clostridial    toxin enzymatic domain.-   5. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the enhanced targeting domain, the    Clostridial toxin enzymatic domain, the protease cleavage site, the    Clostridial toxin translocation domain and the translocation    facilitating domain.-   6. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin translocation    domain, the translocation facilitating domain, the protease cleavage    site, the Clostridial toxin enzymatic domain and the enhanced    targeting domain.-   7. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin translocation    domain, the translocation facilitating domain, the protease cleavage    site, the enhanced targeting domain and the Clostridial toxin    enzymatic domain.-   8. The modified Clostridial toxin according to 1, wherein the    Clostridial toxin enzymatic domain is selected from the group    consisting of a BoNT/A enzymatic domain, a BoNT/B enzymatic domain,    a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E    enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G enzymatic    domain and a TeNT enzymatic domain.-   9. The modified Clostridial toxin according to 1, wherein the    Clostridial toxin translocation domain is selected from the group    consisting of a BoNT/A translocation domain, a BoNT/B translocation    domain, a BoNT/C1 translocation domain, a BoNT/D translocation    domain, a BoNT/E translocation domain, a BoNT/F translocation    domain, a BoNT/G translocation domain and a TeNT translocation    domain.-   10. The modified Clostridial toxin according to 1, wherein the    translocation facilitating domain is a Clostridial toxin    translocation facilitating domain.-   11. The modified Clostridial toxin according to 10, wherein the    Clostridial toxin translocation facilitating domain is selected from    the group consisting of a BoNT/A translocation facilitating domain,    a BoNT/B translocation facilitating domain, a BoNT/C1 translocation    facilitating domain, a BoNT/D translocation domain, a BoNT/E    translocation facilitating domain, a BoNT/F translocation    facilitating domain, a BoNT/G translocation facilitating domain and    a TeNT translocation facilitating domain.-   12. The modified Clostridial toxin according to 10, wherein the    Clostridial toxin translocation facilitating domain comprises an    amino acid sequence selected from the group consisting of amino    acids 874-1110 of SEQ ID NO: 1, amino acids 861-1097 of SEQ ID NO:    2, amino acids 869-1111 of SEQ ID NO: 3, amino acids 865-1098 of SEQ    ID NO: 4, amino acids 848-1085 of SEQ ID NO: 5, amino acids 867-1105    of SEQ ID NO: 6, amino acids 866-1105 of SEQ ID NO: 7 and amino acid    882-1127 of SEQ ID NO: 8.-   13. The modified Clostridial toxin according to 1, wherein the    translocation facilitating domain is an enveloped virus fusogenic    peptide domain.-   14. The modified Clostridial toxin according to 13, wherein the    enveloped virus fusogenic peptide domain is selected from the group    consisting of an influenzavirus fusogenic peptide domain, an    alphavirus fusogenic peptide domain, a vesiculovirus fusogenic    peptide domain, a respirovirus fusogenic peptide domain, a    morbillivirus fusogenic peptide domain, an avulavirus fusogenic    peptide domain, a henipavirus fusogenic peptide domain, a    metapneumovirus fusogenic peptide domain and a foamy virus fusogenic    peptide domain.-   15. The modified Clostridial toxin according to 14, wherein the    influenzavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID    NO: 90 and SEQ ID NO: 91.-   16. The modified Clostridial toxin according to 14, wherein the    alphavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID    NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99,    SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ    ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID    NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:    112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116,    SEQ ID NO: 117 and SEQ ID NO: 118.-   17. The modified Clostridial toxin according to 14, wherein the    vesiculovirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID    NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:    126, SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129.-   18. The modified Clostridial toxin according to 14, wherein the    respirovirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID    NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 and SEQ ID NO: 136.-   19. The modified Clostridial toxin according to 14, wherein the    morbillivirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID    NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO:    144, SEQ ID NO: 145 and SEQ ID NO: 146.-   20. The modified Clostridial toxin according to 14, wherein the    avulavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID    NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO:    154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158    and SEQ ID NO: 159.-   21. The modified Clostridial toxin according to 14, wherein the    henipavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 160 and SEQ ID NO: 161.-   22. The modified Clostridial toxin according to 14, wherein the    metapneumovirus fusogenic peptide domain is SEQ ID NO: 162.-   23. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin H_(CC) targeting domain is selected from    the group consisting of a modified BoNT/A H_(CC) targeting domain, a    modified BoNT/B H_(CC) targeting domain, a modified BoNT/C1 H_(CC)    targeting domain, a modified BoNT/D H_(CC) targeting domain, a    modified BoNT/E H_(CC) targeting domain, a modified BoNT/F H_(CC)    targeting domain, a modified BoNT/G H_(CC) targeting domain and a    modified TeNT H_(CC) targeting domain.-   24. The modified Clostridial toxin according to 23, wherein the    modified BoNT/A H_(CC) targeting domain comprises an amino acid    substitution at a position selected from the group consisting of Trp    1101, Gly 1102, Leu 1105, Tyr 1111, Tyr 1112, Gly 1158, Ile 1163,    Asp 1179, Glu 1203, Phe 1252, Ser 1264, Trp 1266, Tyr 1267, Gln    1270, Gly 1279 and Trp 1282 of SEQ ID NO: 1, and any combination    thereof;    -   wherein the amino acid substitution enhances the binding        activity of the modified BoNT/A H_(CC) targeting domain for a        BoNT/A receptor.-   25. The modified Clostridial toxin according to 23, wherein the    modified BoNT/B H_(CC) targeting domain comprises an amino acid    substitution at a position selected from the group consisting of Trp    1088, Gly 1089, Leu 1092, Tyr 1098, Tyr 1099, Gly 1142, Ile 1147,    Asp 1165, Glu 1191, Ile 1240, Ser 1260, Trp 1262, Tyr 1263, Glu    1266, Gly 1277 and Trp 1280 of SEQ ID NO: 2, and any combination    thereof;    -   wherein the amino acid substitution enhances the binding        activity of the modified BoNT/B H_(CC) targeting domain for a        BoNT/B receptor.-   26. The modified Clostridial toxin according to 23, wherein the    modified BoNT/C1 H_(CC) targeting domain comprises an amino acid    substitution at a position selected from the group consisting of Trp    1102, Gly 1103, Leu 1106, Tyr 1112, Tyr 1113, Gly 1145, Ile 1150,    Asp 1166, Glu 1196, Ile 1247, Gly 1256, Trp 1258, Tyr 1259, His    1261, Gly 1281 and Trp 1284 of SEQ ID NO: 3, and any combination    thereof;    -   wherein the amino acid substitution enhances the binding        activity of the modified BoNT/C1 H_(CC) targeting domain for a        BoNT/C1 receptor.-   27. The modified Clostridial toxin according to 23, wherein the    modified BoNT/D H_(CC) targeting domain comprises an amino acid    substitution at a position selected from the group consisting of Trp    1089, Gly 1090, Leu 1093, Tyr 1099, Tyr 1100, Gly 1132, Ile 1137,    Asp 1153, Asn 1186, Lys 1236, Trp 1238, Arg 1239, Phe 1242, Ser 1262    and Trp 1265 of SEQ ID NO: 4, and any combination thereof;    -   wherein the amino acid substitution enhances the binding        activity of the modified BoNT/D H_(CC) targeting domain for a        BoNT/D receptor.-   28. The modified Clostridial toxin according to 23, wherein the    modified BoNT/E H_(CC) targeting domain comprises an amino acid    substitution at a position selected from the group consisting of Trp    1076, Gly 1077, Leu 1080, Tyr 1086, Tyr 1087, Gly 1124, Ile 1129,    Asp 1146, Glu 1172, Phe 1213, Ser 1221, Trp 1223, Tyr 1224, His    1227, Gly 1236 and Trp 1239 of SEQ ID NO: 5, and any combination    thereof;    -   wherein the amino acid substitution enhances the binding        activity of the modified BoNT/E H_(CC) targeting domain for a        BoNT/E receptor.-   29. The modified Clostridial toxin according to 23, wherein the    modified BoNT/F H_(CC) targeting domain comprises an amino acid    substitution at a position selected from the group consisting of Trp    1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1147, Ile 1152,    Asp 1171, Glu 1195, Phe 1237, Ser 1245, Trp 1247, Tyr 1248, Asn    1251, Gly 1260 and Trp 1263 of SEQ ID NO: 6, and any combination    thereof;    -   wherein the amino acid substitution enhances the binding        activity of the modified BoNT/F H_(CC) targeting domain for a        BoNT/F receptor.-   30. The modified Clostridial toxin according to 23, wherein the    modified BoNT/G H_(CC) targeting domain comprises an amino acid    substitution at a position selected from the group consisting of Trp    1096, Gly 1097, Leu 1100, Tyr 1106, Tyr 1107, Gly 1148, Ile 1153,    Asp 1172, Gln 1198, Ile 1245, Ser 1266, Trp 1268, Tyr 1269, Arg    1272, Gly 1283 and Trp 1285 of SEQ ID NO: 7, and any combination    thereof;    -   wherein the amino acid substitution enhances the binding        activity of the modified BoNT/G H_(CC) targeting domain for a        BoNT/G receptor.-   31. The modified Clostridial toxin according to 23, wherein the    modified TeNT H_(CC) targeting domain comprises an amino acid    substitution at a position selected from the group consisting of Trp    1118, Gly 1119, Leu 1122, Tyr 1128, Tyr 1129, Gly 1172, Ile 1177,    Asp 1194, Asp 1222, Thr 1270, Ser 1287, Trp 1289, Tyr 1290, His    1293, Gly 1300 and Trp 1303 of SEQ ID NO: 8, and any combination    thereof;    -   wherein the amino acid substitution enhances the binding        activity of the modified TeNT H_(CC) targeting domain for a TeNT        receptor.-   32. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin H_(CC) targeting domain comprises at    least one amino acid substitution in a β-trefoil domain.-   33. The modified Clostridial toxin according to 31, wherein the    amino acid substitution is located within an α-fold, a β-fold or a    γ-fold of the β-trefoil domain.-   34. The modified Clostridial toxin according to 31, wherein the    amino acid substitution is located within a β4/β5 β-hairpin turn or    a β8/β9 β-hairpin turn of the β-trefoil domain.-   35. The modified Clostridial toxin according to 33, wherein the    amino acid substitution is a glycine or a phenylalanine.-   36. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin H_(CC) targeting domain comprises a    substitution of a Clostridial toxin H_(CC) targeting domain α-fold    for an α-fold selected from the group consisting of a Clostridial    botulinum serotype A HA-331α-fold, a Clostridial botulinum serotype    B HA-33 1α-fold, a Clostridial botulinum serotype C1 HA-33 1α-fold,    a Clostridial botulinum serotype D HA-33 1α-fold, a Clostridial    botulinum serotype A HA-33 2α-fold, a Clostridial botulinum serotype    B HA-33 2α-fold, a Clostridial botulinum serotype C1 HA-33 2α-fold,    a Clostridial botulinum serotype D HA-33 2α-fold, a Clostridial    botulinum serotype A HA-17 α-fold, a Clostridial botulinum serotype    B HA-17 α-fold, a Clostridial botulinum serotype C1 HA-17 α-fold, a    Clostridial botulinum serotype D HA-17 α-fold, a Clostridial    botulinum serotype A NTNH α-fold, a Clostridial botulinum serotype B    NTNH α-fold, a Clostridial botulinum serotype C1 NTNH α-fold, a    Clostridial botulinum serotype D NTNH α-fold, a Clostridial    botulinum serotype E NTNH α-fold, a Clostridial botulinum serotype F    NTNH α-fold, a Clostridial botulinum serotype G NTNH α-fold, a FGF-1    α-fold, a FGF-2 α-fold, a FGF-4 α-fold, a FGF-8 α-fold, a FGF-9    α-fold, a FGF-17 α-fold and a FGF-18 α-fold.-   37. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin H_(CC) targeting domain comprises a    substitution of a Clostridial toxin H_(CC) targeting domain β-fold    for a β-fold selected from the group consisting of a Clostridial    botulinum serotype A HA-33 1β-fold, a Clostridial botulinum serotype    B HA-33 1β-fold, a Clostridial botulinum serotype C1 HA-33 1β-fold,    a Clostridial botulinum serotype D HA-33 1β-fold, a Clostridial    botulinum serotype A HA-33 2β-fold, a Clostridial botulinum serotype    B HA-33 2β-fold, a Clostridial botulinum serotype C1 HA-33 2β-fold,    a Clostridial botulinum serotype D HA-33 2β-fold, a Clostridial    botulinum serotype A HA-17 β-fold, a Clostridial botulinum serotype    B HA-17 β-fold, a Clostridial botulinum serotype C1 HA-17 β-fold, a    Clostridial botulinum serotype D HA-17 β-fold, a Clostridial    botulinum serotype A NTNH β-fold, a Clostridial botulinum serotype B    NTNH β-fold, a Clostridial botulinum serotype C1 NTNH β-fold, a    Clostridial botulinum serotype D NTNH β-fold, a Clostridial    botulinum serotype E NTNH β-fold, a Clostridial botulinum serotype F    NTNH β-fold, a Clostridial botulinum serotype G NTNH β-fold, a FGF-1    β-fold, a FGF-2 fold, a FGF-4 β-fold, a FGF-8 β-fold, a FGF-9    β-fold, a FGF-17 β-fold and a FGF-18 β-fold.-   38. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin H_(CC) targeting domain comprises a    substitution of a Clostridial toxin H_(CC) targeting domain γ-fold    for a γ-fold selected from the group consisting of a Clostridial    botulinum serotype A HA-33 1γ-fold, a Clostridial botulinum serotype    B HA-33 1γ-fold, a Clostridial botulinum serotype C1 HA-33 1γ-fold,    a Clostridial botulinum serotype D HA-33 1γ-fold, a Clostridial    botulinum serotype A HA-33 2γ-fold, a Clostridial botulinum serotype    B HA-33 2γ-fold, a Clostridial botulinum serotype C1 HA-33 2γ-fold,    a Clostridial botulinum serotype D HA-33 2γ-fold, a Clostridial    botulinum serotype A HA-17 γ-fold, a Clostridial botulinum serotype    B HA-17 γ-fold, a Clostridial botulinum serotype C1 HA-17 γ-fold, a    Clostridial botulinum serotype D HA-17 γ-fold, a Clostridial    botulinum serotype A NTNH γ-fold, a Clostridial botulinum serotype B    NTNH γ-fold, a Clostridial botulinum serotype C1 NTNH γ-fold, a    Clostridial botulinum serotype D NTNH γ-fold, a Clostridial    botulinum serotype E NTNH γ-fold, a Clostridial botulinum serotype F    NTNH γ-fold, a Clostridial botulinum serotype G NTNH γ-fold, a FGF-1    γ-fold, a FGF-2 γ-fold, a FGF-4 γ-fold, a FGF-8 γ-fold, a FGF-9    γ-fold, a FGF-17 γ-fold and a FGF-18 γ-fold.-   39. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin H_(CC) targeting domain comprises a    substitution of a Clostridial toxin H_(CC) targeting domain β4/β5    β-hairpin turn for a β4/β5 β-hairpin turn selected from the group    consisting of a Clostridial botulinum serotype A HA-33 β4/β5    β-hairpin turn, a Clostridial botulinum serotype B HA-33 1β4/β5    β-hairpin turn, a Clostridial botulinum serotype C1 HA-33 1β4/β5    β-hairpin turn, a Clostridial botulinum serotype D HA-33 1β4/β5    β-hairpin turn, a Clostridial botulinum serotype A HA-33 2β4/β5    β-hairpin turn, a Clostridial botulinum serotype B HA-33 2β4/β5    β-hairpin turn, a Clostridial botulinum serotype C1 HA-33 2β4/β5    β-hairpin turn, a Clostridial botulinum serotype D HA-33 2β4/β5    β-hairpin turn, a Clostridial botulinum serotype A HA-17 β4/β5    β-hairpin turn, a Clostridial botulinum serotype B HA-17 β4/β5    β-hairpin turn, a Clostridial botulinum serotype C1 HA-17 β4/β5    β-hairpin turn, a Clostridial botulinum serotype D HA-17 β4/β5    β-hairpin turn, a Clostridial botulinum serotype A NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype B NTNH    β4/β5β-hairpin turn, a Clostridial botulinum serotype C1 NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype D NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype E NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype F NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype G NTNH β4/β5    β-hairpin turn, a FGF-1 β4/β5 β-hairpin turn, a FGF-2 β4/β5    β-hairpin turn, a FGF-4 β4/β5 β-hairpin turn, a FGF-8 β4/β5    β-hairpin turn, a FGF-9 β4/β5 β-hairpin turn, a FGF-17 β4/β5    β-hairpin turn and a FGF-18 β4/β5 β-hairpin turn.-   40. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin H_(CC) targeting domain comprises a    substitution of a Clostridial toxin H_(CC) targeting domain β8/β9    β-hairpin turn for a β8/β9 β-hairpin turn selected from the group    consisting of a Clostridial botulinum serotype A HA-33 β8/β9    β-hairpin turn, a Clostridial botulinum serotype B HA-33 1β8/β9    β-hairpin turn, a Clostridial botulinum serotype C1 HA-33 1β8/β9    β-hairpin turn, a Clostridial botulinum serotype D HA-33 β8/β9    β-hairpin turn, a Clostridial botulinum serotype A HA-33 2β8/β9    β-hairpin turn, a Clostridial botulinum serotype B HA-33 2β8/β9    β-hairpin turn, a Clostridial botulinum serotype C1 HA-33 2β8/β9    β-hairpin turn, a Clostridial botulinum serotype D HA-33 2β8/β9    β-hairpin turn, a Clostridial botulinum serotype A HA-17 β8/β9    β-hairpin turn, a Clostridial botulinum serotype B HA-17 β8/β9    β-hairpin turn, a Clostridial botulinum serotype C1 HA-17 β8/β9    β-hairpin turn, a Clostridial botulinum serotype D HA-17 β8/β9    β-hairpin turn, a Clostridial botulinum serotype A NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype B NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype C1 NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype D NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype E NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype F NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype G NTNH β8/β9    β-hairpin turn, a FGF-1 β8/β9 β-hairpin turn, a FGF-2 β8/β9    β-hairpin turn, a FGF-4 β8/β9 β-hairpin turn, a FGF-8 β8/β9    β-hairpin turn, a FGF-9 β8/β9 β-hairpin turn, a FGF-17 β8/β9    β-hairpin turn and a FGF-18 β8/β9 β-hairpin turn.-   41. The modified Clostridial toxin according to 1, wherein the    protease cleavage site is an endogenous Clostridial toxin di-chain    loop protease cleavage site or an exogenous cleavage site.-   42. The modified Clostridial toxin according to 41, wherein the    endogenous Clostridial toxin di-chain loop protease cleavage site is    selected from the group consisting of a BoNT/A di-chain loop    protease cleavage site, a BoNT/B di-chain loop protease cleavage    site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/D    di-chain loop protease cleavage site, a BoNT/E di-chain loop    protease cleavage site, a BoNT/F di-chain loop protease cleavage    site, a BoNT/G di-chain loop protease cleavage site and a TeNT    di-chain loop protease cleavage site.-   43. The modified Clostridial toxin according to 41, wherein the    exogenous protease cleavage site is selected from the group    consisting of an enterokinase cleavage site, a Thrombin cleavage    site, a Factor Xa cleavage site, a human rhinovirus 3C protease    cleavage site, a tobacco etch virus protease cleavage site, a    dipeptidyl aminopeptidase cleavage site, a small ubiquitin-like    modifier (SUMO)/ubiquitin-like protein-1 (ULP-1) protease cleavage    site, and a Clostridial toxin substrate cleavage site.-   44. The modified Clostridial toxin according to 43, wherein the    Clostridial toxin substrate cleavage site is selected from the group    consisting of a BoNT/A substrate cleavage site, a BoNT/B substrate    cleavage site, a BoNT/C1 substrate cleavage site, a BoNT/D substrate    cleavage site, a BoNT/E substrate cleavage site, a BoNT/F substrate    cleavage site, a BoNT/G substrate cleavage site and a TeNT substrate    cleavage site.-   45. A modified Clostridial toxin comprising:    -   a) a Clostridial toxin enzymatic domain capable of executing an        enzymatic target modification step of a Clostridial toxin        intoxication process;    -   b) a Clostridial toxin translocation domain capable of executing        a translocation step of a Clostridial toxin intoxication        process;    -   c) a translocation facilitating domain capable of facilitating a        translocation step of a Clostridial toxin intoxication process;    -   d) an enhanced targeting domain comprising a Clostridial        non-toxin associated protein β-trefoil domain capable of        executing a cell binding step of a Clostridial toxin        intoxication process; and    -   e) a protease cleavage site    -   wherein cleavage of the protease cleavage site converts the        single-chain form of the modified Clostridial toxin into the        di-chain form.-   46. The modified Clostridial toxin according to 45, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin enzymatic domain,    the protease cleavage site, the Clostridial toxin translocation    domain, the translocation facilitating domain and the enhanced    targeting domain.-   47. The modified Clostridial toxin according to 45, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin enzymatic domain,    the protease cleavage site, the enhanced targeting domain, the    Clostridial toxin translocation domain and the translocation    facilitating domain.-   48. The modified Clostridial toxin according to 45, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the enhanced targeting domain, the    Clostridial toxin translocation domain, the translocation    facilitating domain, the protease cleavage site and the Clostridial    toxin enzymatic domain.-   49. The modified Clostridial toxin according to 45, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the enhanced targeting domain, the    Clostridial toxin enzymatic domain, the protease cleavage site, the    Clostridial toxin translocation domain and the translocation    facilitating domain.-   50. The modified Clostridial toxin according to 45, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin translocation    domain, the translocation facilitating domain, the protease cleavage    site, the Clostridial toxin enzymatic domain and the enhanced    targeting domain.-   51. The modified Clostridial toxin according to 45, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin translocation    domain, the translocation facilitating domain, the protease cleavage    site, the enhanced targeting domain and the Clostridial toxin    enzymatic domain.-   52. The modified Clostridial toxin according to 45, wherein the    Clostridial toxin enzymatic domain is selected from the group    consisting of a BoNT/A enzymatic domain, a BoNT/B enzymatic domain,    a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E    enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G enzymatic    domain and a TeNT enzymatic domain.-   53. The modified Clostridial toxin according to 45, wherein the    Clostridial toxin translocation domain is selected from the group    consisting of a BoNT/A translocation domain, a BoNT/B translocation    domain, a BoNT/C1 translocation domain, a BoNT/D translocation    domain, a BoNT/E translocation domain, a BoNT/F translocation    domain, a BoNT/G translocation domain and a TeNT translocation    domain.-   54. The modified Clostridial toxin according to 45, wherein the    translocation facilitating domain is a Clostridial toxin    translocation facilitating domain.-   55. The modified Clostridial toxin according to 54, wherein the    Clostridial toxin translocation facilitating domain is selected from    the group consisting of a BoNT/A translocation facilitating domain,    a BoNT/B translocation facilitating domain, a BoNT/C1 translocation    facilitating domain, a BoNT/D translocation domain, a BoNT/E    translocation facilitating domain, a BoNT/F translocation    facilitating domain, a BoNT/G translocation facilitating domain and    a TeNT translocation facilitating domain.-   56. The modified Clostridial toxin according to 54, wherein the    Clostridial toxin translocation facilitating domain comprises an    amino acid sequence selected from the group consisting of amino    acids 874-1110 of SEQ ID NO: 1, amino acids 861-1097 of SEQ ID NO:    2, amino acids 869-1111 of SEQ ID NO: 3, amino acids 865-1098 of SEQ    ID NO: 4, amino acids 848-1085 of SEQ ID NO: 5, amino acids 867-1105    of SEQ ID NO: 6, amino acids 866-1105 of SEQ ID NO: 7 and amino acid    882-1127 of SEQ ID NO: 8.-   57. The modified Clostridial toxin according to 45, wherein the    translocation facilitating domain is an enveloped virus fusogenic    peptide domain.-   58. The modified Clostridial toxin according to 57, wherein the    enveloped virus fusogenic peptide domain is selected from the group    consisting of an influenzavirus fusogenic peptide domain, an    alphavirus fusogenic peptide domain, a vesiculovirus fusogenic    peptide domain, a respirovirus fusogenic peptide domain, a    morbillivirus fusogenic peptide domain, an avulavirus fusogenic    peptide domain, a henipavirus fusogenic peptide domain, a    metapneumovirus fusogenic peptide domain and a foamy virus fusogenic    peptide domain.-   59. The modified Clostridial toxin according to 58, wherein the    influenzavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID    NO: 90 and SEQ ID NO: 91.-   60. The modified Clostridial toxin according to 58, wherein the    alphavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID    NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99,    SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ    ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID    NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:    112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116,    SEQ ID NO: 117 and SEQ ID NO: 118.-   61. The modified Clostridial toxin according to 58, wherein the    vesiculovirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID    NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:    126, SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129.-   62. The modified Clostridial toxin according to 58, wherein the    respirovirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID    NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 and SEQ ID NO: 136.-   63. The modified Clostridial toxin according to 58, wherein the    morbillivirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID    NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO:    144, SEQ ID NO: 145 and SEQ ID NO: 146.-   64. The modified Clostridial toxin according to 58, wherein the    avulavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID    NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO:    154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158    and SEQ ID NO: 159.-   65. The modified Clostridial toxin according to 58, wherein the    henipavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 160 and SEQ ID NO: 161.-   66. The modified Clostridial toxin according to 58, wherein the    metapneumovirus fusogenic peptide domain is SEQ ID NO: 162.-   67. The modified Clostridial toxin according to 45, wherein the    Clostridial non-toxin associated protein β-trefoil domain is    selected from the group consisting of a Clostridial botulinum    serotype A HA-33 β-trefoil domain, a Clostridial botulinum serotype    B HA-33 β-trefoil domain, a Clostridial botulinum serotype C1 HA-33    β-trefoil domain, a Clostridial botulinum serotype D HA-33 β-trefoil    domain, a Clostridial botulinum serotype A HA-17 β-trefoil domain, a    Clostridial botulinum serotype B HA-17 β-trefoil domain, a    Clostridial botulinum serotype C1 HA-17 β-trefoil domain, a    Clostridial botulinum serotype D HA-17 β-trefoil domain, a    Clostridial botulinum serotype A NTNH β-trefoil domain, a    Clostridial botulinum serotype B NTNH β-trefoil domain, a    Clostridial botulinum serotype C1 NTNH trefoil domain, a Clostridial    botulinum serotype D NTNH β-trefoil domain, a Clostridial botulinum    serotype E NTNH β-trefoil domain, a Clostridial botulinum serotype F    NTNH β-trefoil domain and a Clostridial botulinum serotype G NTNH    β-trefoil domain.-   68. The modified Clostridial toxin according to 45, wherein the    Clostridial non-toxin associated protein β-trefoil domain is    selected from the group consisting of amino acids 10-144 of SEQ ID    NO: 9, amino acids 151-293 of SEQ ID NO: 9, amino acids 10-144 of    SEQ ID NO: 10, amino acids 151-293 of SEQ ID NO: 10, amino acids    10-144 of SEQ ID NO: 11, amino acids 151-293 of SEQ ID NO: 11, amino    acids 10-146 of SEQ ID NO: 12, amino acids 153-294 of SEQ ID NO: 12,    amino acids 10-144 of SEQ ID NO: 13, amino acids 151-279 of SEQ ID    NO: 13, amino acids 10-144 of SEQ ID NO: 14, amino acids 151-292 of    SEQ ID NO: 14, amino acids 10-146 of SEQ ID NO: 15, amino acids    153-291 of SEQ ID NO: 15, amino acids 10-141 of SEQ ID NO: 16, amino    acids 148-285 of SEQ ID NO: 15, amino acids 10-141 of SEQ ID NO: 16,    amino acids 148-285 of SEQ ID NO: 16, amino acids 10-141 of SEQ ID    NO: 17, amino acids 148-286 of SEQ ID NO: 17, amino acids 10-141 of    SEQ ID NO: 18, amino acids 148-286 of SEQ ID NO: 18, amino acids    9-146 of SEQ ID NO: 19, amino acids 9-146 of SEQ ID NO: 20, amino    acids 9-146 of SEQ ID NO: 21, amino acids 9-146 of SEQ ID NO: 22,    amino acids 1050-1194 of SEQ ID NO: 23, amino acids 1050-1199 of SEQ    ID NO: 24, amino acids 1050-1194 of SEQ ID NO: 25, amino acids    1049-1198 of SEQ ID NO: 26, amino acids 1049-1197 of SEQ ID NO: 27,    amino acids 1049-1197 of SEQ ID NO: 28, amino acids 1014-1163 of SEQ    ID NO: 29, amino acids 1016-1160 of SEQ ID NO: 30, amino acids    1017-1166 of SEQ ID NO: 31 and amino acids 1050-1197 of SEQ ID NO:    1199.-   69. The modified Clostridial toxin according to 45, wherein the    protease cleavage site is an endogenous Clostridial toxin di-chain    loop protease cleavage site or an exogenous cleavage site.-   70. The modified Clostridial toxin according to 69, wherein the    endogenous Clostridial toxin di-chain loop protease cleavage site is    selected from the group consisting of a BoNT/A di-chain loop    protease cleavage site, a BoNT/B di-chain loop protease cleavage    site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/D    di-chain loop protease cleavage site, a BoNT/E di-chain loop    protease cleavage site, a BoNT/F di-chain loop protease cleavage    site, a BoNT/G di-chain loop protease cleavage site and a TeNT    di-chain loop protease cleavage site.-   71. The modified Clostridial toxin according to 69, wherein the    exogenous protease cleavage site is selected from the group    consisting of an enterokinase cleavage site, a Thrombin cleavage    site, a Factor Xa cleavage site, a human rhinovirus 3C protease    cleavage site, a tobacco etch virus protease cleavage site, a    dipeptidyl aminopeptidase cleavage site, a small ubiquitin-like    modifier (SUMO)/ubiquitin-like protein-1(ULP-1) protease cleavage    site, and a Clostridial toxin substrate cleavage site.-   72. The modified Clostridial toxin according to 71, wherein the    Clostridial toxin substrate cleavage site is selected from the group    consisting of a BoNT/A substrate cleavage site, a BoNT/B substrate    cleavage site, a BoNT/C1 substrate cleavage site, a BoNT/D substrate    cleavage site, a BoNT/E substrate cleavage site, a BoNT/F substrate    cleavage site, a BoNT/G substrate cleavage site and a TeNT substrate    cleavage site.-   73. A modified Clostridial toxin comprising:    -   a) a Clostridial toxin enzymatic domain capable of executing an        enzymatic target modification step of a Clostridial toxin        intoxication process;    -   b) a Clostridial toxin translocation domain capable of executing        a translocation step of a Clostridial toxin intoxication        process;    -   c) a translocation facilitating domain capable of facilitating a        translocation step of a Clostridial toxin intoxication process;    -   d) an enhanced targeting domain comprising a FGF β-trefoil        domain capable of selectively binding an FGFR3; and    -   e) a protease cleavage site    -   wherein cleavage of the protease cleavage site converts the        single-chain form of the modified Clostridial toxin into the        di-chain form.-   74. The modified Clostridial toxin according to 73, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin enzymatic domain,    the protease cleavage site, the Clostridial toxin translocation    domain, the translocation facilitating domain and the enhanced    targeting domain.-   75. The modified Clostridial toxin according to 73, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin enzymatic domain,    the protease cleavage site, the enhanced targeting domain, the    Clostridial toxin translocation domain and the translocation    facilitating domain.-   76. The modified Clostridial toxin according to 73, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the enhanced targeting domain, the    Clostridial toxin translocation domain, the translocation    facilitating domain, the protease cleavage site and the Clostridial    toxin enzymatic domain.-   77. The modified Clostridial toxin according to 73, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the enhanced targeting domain, the    Clostridial toxin enzymatic domain, the protease cleavage site, the    Clostridial toxin translocation domain and the translocation    facilitating domain.-   78. The modified Clostridial toxin according to 73, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin translocation    domain, the translocation facilitating domain, the protease cleavage    site, the Clostridial toxin enzymatic domain and the enhanced    targeting domain.-   79. The modified Clostridial toxin according to 73, wherein the    modified Clostridial toxin comprises, in a linear amino-to-carboxyl    single polypeptide order, the Clostridial toxin translocation    domain, the translocation facilitating domain, the protease cleavage    site, the enhanced targeting domain and the Clostridial toxin    enzymatic domain.-   80. The modified Clostridial toxin according to 73, wherein the    Clostridial toxin enzymatic domain is selected from the group    consisting of a BoNT/A enzymatic domain, a BoNT/B enzymatic domain,    a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E    enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G enzymatic    domain and a TeNT enzymatic domain.-   81. The modified Clostridial toxin according to 73, wherein the    Clostridial toxin translocation domain is selected from the group    consisting of a BoNT/A translocation domain, a BoNT/B translocation    domain, a BoNT/C1 translocation domain, a BoNT/D translocation    domain, a BoNT/E translocation domain, a BoNT/F translocation    domain, a BoNT/G translocation domain and a TeNT translocation    domain.-   82. The modified Clostridial toxin according to 73, wherein the    translocation facilitating domain is a Clostridial toxin    translocation facilitating domain.-   83. The modified Clostridial toxin according to 82, wherein the    Clostridial toxin translocation facilitating domain is selected from    the group consisting of a BoNT/A translocation facilitating domain,    a BoNT/B translocation facilitating domain, a BoNT/C1 translocation    facilitating domain, a BoNT/D translocation domain, a BoNT/E    translocation facilitating domain, a BoNT/F translocation    facilitating domain, a BoNT/G translocation facilitating domain and    a TeNT translocation facilitating domain.-   84. The modified Clostridial toxin according to 82, wherein the    Clostridial toxin translocation facilitating domain comprises an    amino acid sequence selected from the group consisting of amino    acids 874-1110 of SEQ ID NO: 1, amino acids 861-1097 of SEQ ID NO:    2, amino acids 869-1111 of SEQ ID NO: 3, amino acids 865-1098 of SEQ    ID NO: 4, amino acids 848-1085 of SEQ ID NO: 5, amino acids 867-1105    of SEQ ID NO: 6, amino acids 866-1105 of SEQ ID NO: 7 and amino acid    882-1127 of SEQ ID NO: 8.-   85. The modified Clostridial toxin according to 73, wherein the    translocation facilitating domain is an enveloped virus fusogenic    peptide domain.-   86. The modified Clostridial toxin according to 85, wherein the    enveloped virus fusogenic peptide domain is selected from the group    consisting of an influenzavirus fusogenic peptide domain, an    alphavirus fusogenic peptide domain, a vesiculovirus fusogenic    peptide domain, a respirovirus fusogenic peptide domain, a    morbillivirus fusogenic peptide domain, an avulavirus fusogenic    peptide domain, a henipavirus fusogenic peptide domain, a    metapneumovirus fusogenic peptide domain and a foamy virus fusogenic    peptide domain.-   87. The modified Clostridial toxin according to 86, wherein the    influenzavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID    NO: 90 and SEQ ID NO: 91.-   88. The modified Clostridial toxin according to 86, wherein the    alphavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID    NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99,    SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ    ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID    NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:    112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116,    SEQ ID NO: 117 and SEQ ID NO: 118.-   89. The modified Clostridial toxin according to 86, wherein the    vesiculovirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID    NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:    126, SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129.-   90. The modified Clostridial toxin according to 86, wherein the    respirovirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID    NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 and SEQ ID NO: 136.-   91. The modified Clostridial toxin according to 86, wherein the    morbillivirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID    NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO:    144, SEQ ID NO: 145 and SEQ ID NO: 146.-   92. The modified Clostridial toxin according to 86, wherein the    avulavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID    NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO:    154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158    and SEQ ID NO: 159.-   93. The modified Clostridial toxin according to 86, wherein the    henipavirus fusogenic peptide domain is selected from the group    consisting of SEQ ID NO: 160 and SEQ ID NO: 161.-   94. The modified Clostridial toxin according to 86, wherein the    metapneumovirus fusogenic peptide domain is SEQ ID NO: 162.-   95. The modified Clostridial toxin according to 73, wherein the FGF    β-trefoil domain is selected from the group consisting of a FGF-1    β-trefoil domain, a FGF-2 β-trefoil domain, a FGF-4 β-trefoil    domain, a FGF-8 β-trefoil domain, a FGF-9 β-trefoil domain, a FGF-17    β-trefoil domain and a FGF-18 β-trefoil domain.-   96. The modified Clostridial toxin according to 73, wherein the FGF    β-trefoil domain is selected from the group consisting of amino    acids 26-155 of SEQ ID NO: 33, amino acids 29-155 of SEQ ID NO: 34,    amino acids 83-206 of SEQ ID NO: 35, amino acids 43-172 of SEQ ID    NO: 36, amino acids 63-196 of SEQ ID NO: 37, amino acids 55-183 of    SEQ ID NO: 38 and amino acids 54-183 of SEQ ID NO: 39.-   97. The modified Clostridial toxin according to 73, wherein the    protease cleavage site is an endogenous Clostridial toxin di-chain    loop protease cleavage site or an exogenous cleavage site.-   98. The modified Clostridial toxin according to 97, wherein the    endogenous Clostridial toxin di-chain loop protease cleavage site is    selected from the group consisting of a BoNT/A di-chain loop    protease cleavage site, a BoNT/B di-chain loop protease cleavage    site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/D    di-chain loop protease cleavage site, a BoNT/E di-chain loop    protease cleavage site, a BoNT/F di-chain loop protease cleavage    site, a BoNT/G di-chain loop protease cleavage site and a TeNT    di-chain loop protease cleavage site.-   99. The modified Clostridial toxin according to 97, wherein the    exogenous protease cleavage site is selected from the group    consisting of an enterokinase cleavage site, a Thrombin cleavage    site, a Factor Xa cleavage site, a human rhinovirus 3C protease    cleavage site, a tobacco etch virus protease cleavage site, a    dipeptidyl aminopeptidase cleavage site, a small ubiquitin-like    modifier (SUMO)/ubiquitin-like protein-1(ULP-1) protease cleavage    site, and a Clostridial toxin substrate cleavage site.-   100. The modified Clostridial toxin according to 99, wherein the    Clostridial toxin substrate cleavage site is selected from the group    consisting of a BoNT/A substrate cleavage site, a BoNT/B substrate    cleavage site, a BoNT/C1 substrate cleavage site, a BoNT/D substrate    cleavage site, a BoNT/E substrate cleavage site, a BoNT/F substrate    cleavage site, a BoNT/G substrate cleavage site and a TeNT substrate    cleavage site.-   101. A polynucleotide molecule encoding a modified Clostridial    toxin, the polynucleotide molecule comprising:    -   a) a polynucleotide molecule encoding a Clostridial toxin        enzymatic domain capable of executing an enzymatic target        modification step of a Clostridial toxin intoxication process;    -   b) a polynucleotide molecule encoding a Clostridial toxin        translocation domain capable of executing a translocation step        of a Clostridial toxin intoxication process;    -   c) a polynucleotide molecule encoding a translocation        facilitating domain capable of facilitating a translocation step        of a Clostridial toxin intoxication process;    -   d) a polynucleotide molecule encoding an enhanced targeting        domain comprising a modified Clostridial H_(CC) targeting domain        capable of executing a cell binding step of a Clostridial toxin        intoxication process; and    -   e) a polynucleotide molecule encoding a protease cleavage site    -   wherein cleavage of the protease cleavage site converts the        single-chain form of the modified Clostridial toxin into the        di-chain form; and    -   wherein the modified Clostridial H_(CC) targeting domain        exhibits enhanced binding activity for an endogenous Clostridial        toxin receptor relative to the binding activity of a        naturally-occurring Clostridial H_(CC) targeting domain from        which the modified Clostridial H_(CC) targeting domain is        derived.-   102. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule encodes the modified Clostridial toxin of    any one of claims 1-7.-   103. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule encoding a Clostridial toxin enzymatic    domain is selected from the group consisting of a polynucleotide    molecule encoding a BoNT/A enzymatic domain, a BoNT/B enzymatic    domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a    BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G    enzymatic domain and a TeNT enzymatic domain.-   104. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule encoding a Clostridial toxin translocation    domain is selected from the group consisting of a polynucleotide    molecule encoding a BoNT/A translocation domain, a BoNT/B    translocation domain, a BoNT/C1 translocation domain, a BoNT/D    translocation domain, a BoNT/E translocation domain, a BoNT/F    translocation domain, a BoNT/G translocation domain and a TeNT    translocation domain.-   105. The polynucleotide molecule according to 101, wherein the    translocation facilitating domain is a Clostridial toxin    translocation facilitating domain.-   106. The polynucleotide molecule according to 105, wherein the    Clostridial toxin translocation facilitating domain is selected from    the group consisting of a BoNT/A translocation facilitating domain,    a BoNT/B translocation facilitating domain, a BoNT/C1 translocation    facilitating domain, a BoNT/D translocation domain, a BoNT/E    translocation facilitating domain, a BoNT/F translocation    facilitating domain, a BoNT/G translocation facilitating domain and    a TeNT translocation facilitating domain.-   107. The polynucleotide molecule according to 101, wherein the    translocation facilitating domain is an enveloped virus fusogenic    peptide domain.-   108. The polynucleotide molecule according to 107, wherein the    enveloped virus fusogenic peptide domain is selected from the group    consisting of an influenzavirus fusogenic peptide domain, an    alphavirus fusogenic peptide domain, a vesiculovirus fusogenic    peptide domain, a respirovirus fusogenic peptide domain, a    morbillivirus fusogenic peptide domain, an avulavirus fusogenic    peptide domain, a henipavirus fusogenic peptide domain, a    metapneumovirus fusogenic peptide domain and a foamy virus fusogenic    peptide domain.-   109. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule encoding a modified Clostridial toxin H_(CC)    targeting domain is selected from the group consisting of a    polynucleotide molecule encoding a modified BoNT/A H_(CC) targeting    domain, a modified BoNT/B H_(CC) targeting domain, a modified    BoNT/C1 H_(CC) targeting domain, a modified BoNT/D H_(CC) targeting    domain, a modified BoNT/E H_(CC) targeting domain, a modified BoNT/F    H_(CC) targeting domain, a modified BoNT/G H_(CC) targeting domain    and a modified TeNT H_(CC) targeting domain.-   110. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule encoding a modified Clostridial toxin H_(CC)    targeting domain comprises a polynucleotide molecule encoding a    substitution of a Clostridial toxin H_(CC) targeting domain α-fold    for an α-fold selected from the group consisting of a Clostridial    botulinum serotype A HA-33 1α-fold, a Clostridial botulinum serotype    B HA-33 1α-fold, a Clostridial botulinum serotype C1 HA-33 1α-fold,    a Clostridial botulinum serotype D HA-33 1α-fold, a Clostridial    botulinum serotype A HA-33 2α-fold, a Clostridial botulinum serotype    B HA-33 2α-fold, a Clostridial botulinum serotype C1 HA-33 2α-fold,    a Clostridial botulinum serotype D HA-33 2α-fold, a Clostridial    botulinum serotype A HA-17 α-fold, a Clostridial botulinum serotype    B HA-17 α-fold, a Clostridial botulinum serotype C1 HA-17 α-fold, a    Clostridial botulinum serotype D HA-17 α-fold, a Clostridial    botulinum serotype A NTNH α-fold, a Clostridial botulinum serotype B    NTNH α-fold, a Clostridial botulinum serotype C1 NTNH α-fold, a    Clostridial botulinum serotype D NTNH α-fold, a Clostridial    botulinum serotype E NTNH α-fold, a Clostridial botulinum serotype F    NTNH α-fold, a Clostridial botulinum serotype G NTNH α-fold, a FGF-1    α-fold, a FGF-2 α-fold, a FGF-4 α-fold, a FGF-8 α-fold, a FGF-9    α-fold, a FGF-17 α-fold and a FGF-18 α-fold.-   111. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule encoding a modified Clostridial toxin H_(CC)    targeting domain comprises a polynucleotide molecule encoding a    substitution of a Clostridial toxin H_(CC) targeting domain β-fold    for a β-fold selected from the group consisting of a Clostridial    botulinum serotype A HA-33 1β-fold, a Clostridial botulinum serotype    B HA-33 1β-fold, a Clostridial botulinum serotype C1 HA-33 1β-fold,    a Clostridial botulinum serotype D HA-33 1β-fold, a Clostridial    botulinum serotype A HA-33 2β-fold, a Clostridial botulinum serotype    B HA-33 2β-fold, a Clostridial botulinum serotype C1 HA-33 2β-fold,    a Clostridial botulinum serotype D HA-33 2β-fold, a Clostridial    botulinum serotype A HA-17 β-fold, a Clostridial botulinum serotype    B HA-17 β-fold, a Clostridial botulinum serotype C1 HA-17 β-fold, a    Clostridial botulinum serotype D HA-17 β-fold, a Clostridial    botulinum serotype A NTNH β-fold, a Clostridial botulinum serotype B    NTNH β-fold, a Clostridial botulinum serotype C1 NTNH β-fold, a    Clostridial botulinum serotype D NTNH β-fold, a Clostridial    botulinum serotype E NTNH β-fold, a Clostridial botulinum serotype F    NTNH β-fold, a Clostridial botulinum serotype G NTNH β-fold, a FGF-1    β-fold, a FGF-2 β-fold, a FGF-4 β-fold, a FGF-8 β-fold, a FGF-9    β-fold, a FGF-17 β-fold and a FGF-18 β-fold.-   112. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule encoding a modified Clostridial toxin H_(CC)    targeting domain comprises a polynucleotide molecule encoding a    substitution of a Clostridial toxin H_(CC) targeting domain γ-fold    for a γ-fold selected from the group consisting of a Clostridial    botulinum serotype A HA-33 1γ-fold, a Clostridial botulinum serotype    B HA-33 1γ-fold, a Clostridial botulinum serotype C1 HA-33 1γ-fold,    a Clostridial botulinum serotype D HA-33 1γ-fold, a Clostridial    botulinum serotype A HA-33 2γ-fold, a Clostridial botulinum serotype    B HA-33 2γ-fold, a Clostridial botulinum serotype C1 HA-33 2γ-fold,    a Clostridial botulinum serotype D HA-33 2γ-fold, a Clostridial    botulinum serotype A HA-17 γ-fold, a Clostridial botulinum serotype    B HA-17 γ-fold, a Clostridial botulinum serotype C1 HA-17 γ-fold, a    Clostridial botulinum serotype D HA-17 γ-fold, a Clostridial    botulinum serotype A NTNH γ-fold, a Clostridial botulinum serotype B    NTNH γ-fold, a Clostridial botulinum serotype C1 NTNH γ-fold, a    Clostridial botulinum serotype D NTNH γ-fold, a Clostridial    botulinum serotype E NTNH γ-fold, a Clostridial botulinum serotype F    NTNH γ-fold, a Clostridial botulinum serotype G NTNH γ-fold, a FGF-1    γ-fold, a FGF-2 γ-fold, a FGF-4 γ-fold, a FGF-8 γ-fold, a FGF-9    γ-fold, a FGF-17 γ-fold and a FGF-18 γ-fold.-   113. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule encoding a modified Clostridial toxin H_(CC)    targeting domain comprises a polynucleotide molecule encoding a    substitution of a Clostridial toxin H_(CC) targeting domain β4/β5    β-hairpin turn for a β4/β5 β-hairpin turn selected from the group    consisting of a Clostridial botulinum serotype A HA-33 β4/β5    β-hairpin turn, a Clostridial botulinum serotype B HA-33 1β4/β5    β-hairpin turn, a Clostridial botulinum serotype C1 HA-33 1β4/β5    β-hairpin turn, a Clostridial botulinum serotype D HA-33 1β4/β5    β-hairpin turn, a Clostridial botulinum serotype A HA-33 2β4/β5    β-hairpin turn, a Clostridial botulinum serotype B HA-33 2β4/β5    β-hairpin turn, a Clostridial botulinum serotype C1 HA-33 2β4/β5    β-hairpin turn, a Clostridial botulinum serotype D HA-33 2β4/β5    β-hairpin turn, a Clostridial botulinum serotype A HA-17 β4/β5    β-hairpin turn, a Clostridial botulinum serotype B HA-17 β4/β5    β-hairpin turn, a Clostridial botulinum serotype C1 HA-17 β4/β5    β-hairpin turn, a Clostridial botulinum serotype D HA-17 β4/β5    β-hairpin turn, a Clostridial botulinum serotype A NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype B NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype C1 NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype D NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype E NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype F NTNH β4/β5    β-hairpin turn, a Clostridial botulinum serotype G NTNH β4/β5    β-hairpin turn, a FGF-1 β4/β5 β-hairpin turn, a FGF-2 β4/β5    β-hairpin turn, a FGF-4 β4/β5 β-hairpin turn, a FGF-8 β4/β5    β-hairpin turn, a FGF-9 β4/β5 β-hairpin turn, a FGF-17 β4/β5    β-hairpin turn and a FGF-18 β4/β5 β-hairpin turn.-   114. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule encoding a modified Clostridial toxin H_(CC)    targeting domain comprises a polynucleotide molecule encoding a    substitution of a Clostridial toxin H_(CC) targeting domain β8/β9    β-hairpin turn for a β8/β9 β-hairpin turn selected from the group    consisting of a Clostridial botulinum serotype A HA-33 β8/β9    β-hairpin turn, a Clostridial botulinum serotype B HA-33 1β8/β9    β-hairpin turn, a Clostridial botulinum serotype C1 HA-33 1β8/β9    β-hairpin turn, a Clostridial botulinum serotype D HA-33 1β8/β9    β-hairpin turn, a Clostridial botulinum serotype A HA-33 2β8/β9    β-hairpin turn, a Clostridial botulinum serotype B HA-33 2β8/β9    β-hairpin turn, a Clostridial botulinum serotype C1 HA-33 2β8/β9    β-hairpin turn, a Clostridial botulinum serotype D HA-33 2β8/β9    β-hairpin turn, a Clostridial botulinum serotype A HA-17 β8/β9    β-hairpin turn, a Clostridial botulinum serotype B HA-17 β8/β9    β-hairpin turn, a Clostridial botulinum serotype C1 HA-17 β8/β9    β-hairpin turn, a Clostridial botulinum serotype D HA-17 β8/β9    β-hairpin turn, a Clostridial botulinum serotype A NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype B NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype C1 NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype D NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype E NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype F NTNH β8/β9    β-hairpin turn, a Clostridial botulinum serotype G NTNH β8/β9    β-hairpin turn, a FGF-1 β8/β9 β-hairpin turn, a FGF-2 β8/β9    β-hairpin turn, a FGF-4 β8/β9 β-hairpin turn, a FGF-8 β8/β9    β-hairpin turn, a FGF-9 β8/β9 β-hairpin turn, a FGF-17 β8/β9    β-hairpin turn and a FGF-18 β8/β9 β-hairpin turn.-   115. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule encoding the protease cleavage site is a    polynucleotide molecule encoding an endogenous Clostridial toxin    di-chain loop protease cleavage site or a polynucleotide molecule    encoding an exogenous cleavage site.-   116. The polynucleotide molecule according to 101, wherein the    polynucleotide molecule comprises an expression vector.-   117. A polynucleotide molecule encoding a modified Clostridial    toxin, the polynucleotide molecule comprising:    -   a) a polynucleotide molecule encoding a Clostridial toxin        enzymatic domain capable of executing an enzymatic target        modification step of a Clostridial toxin intoxication process;    -   b) a polynucleotide molecule encoding a Clostridial toxin        translocation domain capable of executing a translocation step        of a Clostridial toxin intoxication process;    -   c) a polynucleotide molecule encoding a translocation        facilitating domain capable of facilitating a translocation step        of a Clostridial toxin intoxication process;    -   d) a polynucleotide molecule encoding an enhanced targeting        domain comprising a Clostridial non-toxin associated protein        β-trefoil domain capable of executing a cell binding step of a        Clostridial toxin intoxication process; and    -   e) a polynucleotide molecule encoding a protease cleavage site    -   wherein cleavage of the protease cleavage site converts the        single-chain form of the modified Clostridial toxin into the        di-chain form.-   118. The polynucleotide molecule according to 115, wherein the    polynucleotide molecule encodes a modified Clostridial toxin of any    one of claims 45-51.-   119. The polynucleotide molecule according to 117, wherein the    polynucleotide molecule encoding a Clostridial toxin enzymatic    domain is selected from the group consisting of a polynucleotide    molecule encoding a BoNT/A enzymatic domain, a BoNT/B enzymatic    domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a    BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G    enzymatic domain and a TeNT enzymatic domain.-   120. The polynucleotide molecule according to 117, wherein the    polynucleotide molecule encoding a Clostridial toxin translocation    domain is selected from the group consisting of a polynucleotide    molecule encoding a BoNT/A translocation domain, a BoNT/B    translocation domain, a BoNT/C1 translocation domain, a BoNT/D    translocation domain, a BoNT/E translocation domain, a BoNT/F    translocation domain, a BoNT/G translocation domain and a TeNT    translocation domain.-   121. The polynucleotide molecule according to 117, wherein the    translocation facilitating domain is a Clostridial toxin    translocation facilitating domain.-   122. The polynucleotide molecule according to 121, wherein the    Clostridial toxin translocation facilitating domain is selected from    the group consisting of a BoNT/A translocation facilitating domain,    a BoNT/B translocation facilitating domain, a BoNT/C1 translocation    facilitating domain, a BoNT/D translocation domain, a BoNT/E    translocation facilitating domain, a BoNT/F translocation    facilitating domain, a BoNT/G translocation facilitating domain and    a TeNT translocation facilitating domain.-   123. The polynucleotide molecule according to 117, wherein the    translocation facilitating domain is an enveloped virus fusogenic    peptide domain.-   124. The polynucleotide molecule according to 123, wherein the    enveloped virus fusogenic peptide domain is selected from the group    consisting of an influenzavirus fusogenic peptide domain, an    alphavirus fusogenic peptide domain, a vesiculovirus fusogenic    peptide domain, a respirovirus fusogenic peptide domain, a    morbillivirus fusogenic peptide domain, an avulavirus fusogenic    peptide domain, a henipavirus fusogenic peptide domain, a    metapneumovirus fusogenic peptide domain and a foamy virus fusogenic    peptide domain.-   125. The polynucleotide molecule according to 117, wherein the    polynucleotide molecule encoding a Clostridial non-toxin associated    protein β-trefoil domain selected from the group consisting of a    polynucleotide molecule encoding a Clostridial botulinum serotype A    HA-33 β-trefoil domain, a Clostridial botulinum serotype B HA-33    β-trefoil domain, a Clostridial botulinum serotype C1 HA-33    β-trefoil domain, a Clostridial botulinum serotype D HA-33 β-trefoil    domain, a Clostridial botulinum serotype A HA-176-trefoil domain, a    Clostridial botulinum serotype B HA-176-trefoil domain, a    Clostridial botulinum serotype C1 HA-176-trefoil domain, a    Clostridial botulinum serotype D HA-17 β-trefoil domain, a    Clostridial botulinum serotype A NTNH β-trefoil domain, a    Clostridial botulinum serotype B NTNH β-trefoil domain, a    Clostridial botulinum serotype C1 NTNH β-trefoil domain, a    Clostridial botulinum serotype D NTNH β-trefoil domain, a    Clostridial botulinum serotype E NTNH β-trefoil domain, a    Clostridial botulinum serotype F NTNH β-trefoil domain and a    Clostridial botulinum serotype G NTNH β-trefoil domain.-   126. The polynucleotide molecule according to 117, wherein the    polynucleotide molecule encoding the protease cleavage site is a    polynucleotide molecule encoding an endogenous Clostridial toxin    di-chain loop protease cleavage site or a polynucleotide molecule    encoding an exogenous cleavage site.-   127. The polynucleotide molecule according to 117, wherein the    polynucleotide molecule comprises an expression vector.-   128. A polynucleotide molecule encoding a modified Clostridial    toxin, the polynucleotide molecule comprising:    -   a) a polynucleotide molecule encoding a Clostridial toxin        enzymatic domain capable of executing an enzymatic target        modification step of a Clostridial toxin intoxication process;    -   b) a polynucleotide molecule encoding a Clostridial toxin        translocation domain capable of executing a translocation step        of a Clostridial toxin intoxication process;    -   c) a polynucleotide molecule encoding a Clostridial toxin        translocation facilitating domain capable of facilitating a        translocation step of a Clostridial toxin intoxication process;    -   d) a polynucleotide molecule encoding an enhanced targeting        domain comprising a FGF β-trefoil domain capable of selectively        binding an FGFR3; and    -   e) a polynucleotide molecule encoding a protease cleavage site    -   wherein cleavage of the protease cleavage site converts the        single-chain form of the modified Clostridial toxin into the        di-chain form.-   129. The polynucleotide molecule according to 128, wherein the    polynucleotide molecule encodes a modified Clostridial toxin of any    one of claims 73-79.-   130. The polynucleotide molecule according to 128, wherein the    polynucleotide molecule encoding a Clostridial toxin enzymatic    domain is selected from the group consisting of a polynucleotide    molecule encoding a BoNT/A enzymatic domain, a BoNT/B enzymatic    domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a    BoNT/E enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G    enzymatic domain and a TeNT enzymatic domain.-   131. The polynucleotide molecule according to 128, wherein the    polynucleotide molecule encoding a Clostridial toxin translocation    domain is selected from the group consisting of a polynucleotide    molecule encoding a BoNT/A translocation domain, a BoNT/B    translocation domain, a BoNT/C1 translocation domain, a BoNT/D    translocation domain, a BoNT/E translocation domain, a BoNT/F    translocation domain, a BoNT/G translocation domain and a TeNT    translocation domain.-   132. The polynucleotide molecule according to 128, wherein the    translocation facilitating domain is a Clostridial toxin    translocation facilitating domain.-   133. The polynucleotide molecule according to 132, wherein the    Clostridial toxin translocation facilitating domain is selected from    the group consisting of a BoNT/A translocation facilitating domain,    a BoNT/B translocation facilitating domain, a BoNT/C1 translocation    facilitating domain, a BoNT/D translocation domain, a BoNT/E    translocation facilitating domain, a BoNT/F translocation    facilitating domain, a BoNT/G translocation facilitating domain and    a TeNT translocation facilitating domain.-   134. The polynucleotide molecule according to 128, wherein the    translocation facilitating domain is an enveloped virus fusogenic    peptide domain.-   135. The polynucleotide molecule according to 134, wherein the    enveloped virus fusogenic peptide domain is selected from the group    consisting of an influenzavirus fusogenic peptide domain, an    alphavirus fusogenic peptide domain, a vesiculovirus fusogenic    peptide domain, a respirovirus fusogenic peptide domain, a    morbillivirus fusogenic peptide domain, an avulavirus fusogenic    peptide domain, a henipavirus fusogenic peptide domain, a    metapneumovirus fusogenic peptide domain and a foamy virus fusogenic    peptide domain.-   136. The polynucleotide molecule according to 128, wherein the    polynucleotide molecule encoding a FGF β-trefoil domain is selected    from the group consisting of a polynucleotide molecule encoding a    FGF-1 β-trefoil domain, a FGF-2 β-trefoil domain, a FGF-4 β-trefoil    domain, a FGF-8 β-trefoil domain, a FGF-9 β-trefoil domain, a FGF-17    β-trefoil domain and a FGF-18 β-trefoil domain.-   137. The polynucleotide molecule according to 128, wherein the    polynucleotide molecule encoding the protease cleavage site is a    polynucleotide molecule encoding an endogenous Clostridial toxin    di-chain loop protease cleavage site or a polynucleotide molecule    encoding an exogenous cleavage site.-   138. The polynucleotide molecule according to 128, wherein the    polynucleotide molecule comprises an expression vector.-   139. A method of producing a modified Clostridial toxin comprising    the step of expressing a polynucleotide molecule encoding a modified    Clostridial toxin in a cell, the polynucleotide molecule comprising:    -   a) a polynucleotide molecule encoding a Clostridial toxin        enzymatic domain capable of executing an enzymatic target        modification step of a Clostridial toxin intoxication process;    -   b) a polynucleotide molecule encoding a Clostridial toxin        translocation domain capable of executing a translocation step        of a Clostridial toxin intoxication process;    -   c) a polynucleotide molecule encoding a translocation        facilitating domain capable of facilitating a translocation step        of a Clostridial toxin intoxication process;    -   d) a polynucleotide molecule encoding an enhanced targeting        domain comprising a modified Clostridial H_(CC) targeting domain        capable of executing a cell binding step of a Clostridial toxin        intoxication process; and    -   e) a polynucleotide molecule encoding a protease cleavage site    -   wherein cleavage of the protease cleavage site converts the        single-chain form of the modified Clostridial toxin into the        di-chain form; and    -   wherein the modified Clostridial H_(CC) targeting domain        exhibits enhanced binding activity for an endogenous Clostridial        toxin receptor relative to the binding activity of a        naturally-occurring Clostridial H_(CC) targeting domain from        which the modified Clostridial binding H_(CC) targeting is        derived.-   140. The method according to 139, wherein the polynucleotide    molecule is any one of the polynucleotide molecules of 102.-   141. A method of producing a modified Clostridial toxin comprising    the step of expressing a polynucleotide molecule encoding a modified    Clostridial toxin in a cell, the polynucleotide molecule comprising:    -   a) a polynucleotide molecule encoding a Clostridial toxin        enzymatic domain capable of executing an enzymatic target        modification step of a Clostridial toxin intoxication process;    -   b) a polynucleotide molecule encoding a Clostridial toxin        translocation domain capable of executing a translocation step        of a Clostridial toxin intoxication process;    -   c) a polynucleotide molecule encoding a translocation        facilitating domain capable of facilitating a translocation step        of a Clostridial toxin intoxication process;    -   d) a polynucleotide molecule encoding an enhanced targeting        domain comprising a Clostridial non-toxin associated protein        β-trefoil domain capable of executing a cell binding step of a        Clostridial toxin intoxication process; and    -   e) a polynucleotide molecule encoding a protease cleavage site    -   wherein cleavage of the protease cleavage site converts the        single-chain form of the modified Clostridial toxin into the        di-chain form.-   142. The method according to 141, wherein the polynucleotide    molecule is any one of the polynucleotide molecules of 118.-   143. A method of producing a modified Clostridial toxin comprising    the step of expressing a polynucleotide molecule encoding a modified    Clostridial toxin in a cell, the polynucleotide molecule comprising:    -   a) a polynucleotide molecule encoding a Clostridial toxin        enzymatic domain capable of executing an enzymatic target        modification step of a Clostridial toxin intoxication process;    -   b) a polynucleotide molecule encoding a Clostridial toxin        translocation domain capable of executing a translocation step        of a Clostridial toxin intoxication process;    -   c) a polynucleotide molecule encoding a translocation        facilitating domain capable of facilitating a translocation step        of a Clostridial toxin intoxication process;    -   d) a polynucleotide molecule encoding an enhanced targeting        domain comprising a FGF β-trefoil domain capable of selectively        binding an FGFR3; and    -   e) a polynucleotide molecule encoding a protease cleavage site    -   wherein cleavage of the protease cleavage site converts the        single-chain form of the modified Clostridial toxin into the        di-chain form.-   144. The method according to 143, wherein the polynucleotide    molecule is any one of the polynucleotide molecules of 129.-   145. A method of producing a modified Clostridial toxin comprising    the steps of:    -   a) introducing into a cell a polynucleotide molecule encoding a        modified Clostridial toxin, the polynucleotide molecule        comprising:        -   i) a polynucleotide molecule encoding a Clostridial toxin            enzymatic domain capable of executing an enzymatic target            modification step of a Clostridial toxin intoxication            process;        -   ii) a polynucleotide molecule encoding a Clostridial toxin            translocation domain capable of executing a translocation            step of a Clostridial toxin intoxication process; iii) a            polynucleotide molecule encoding a translocation            facilitating domain capable of facilitating a translocation            step of a Clostridial toxin intoxication process;        -   iv) a polynucleotide molecule encoding an enhanced targeting            domain comprising a modified Clostridial H_(CC) targeting            domain capable of executing a cell binding step of a            Clostridial toxin intoxication process; and        -   v) a polynucleotide molecule encoding a protease cleavage            site        -   wherein cleavage of the protease cleavage site converts the            single-chain form of the modified Clostridial toxin into the            di-chain form; and        -   wherein the modified Clostridial H_(CC) targeting domain            exhibits enhanced binding activity for an endogenous            Clostridial toxin receptor relative to the binding activity            of a naturally-occurring Clostridial H_(CC) targeting domain            from which the modified Clostridial H_(CC) targeting domain            is derived.    -   b) expressing the modified Clostridial toxin encoded by the        polynucleotide molecule.-   146. The method according to 145, wherein the polynucleotide    molecule is any one of the polynucleotide molecules of 102.-   147. A method of producing a modified Clostridial toxin comprising    the steps of:    -   a) introducing into a cell a polynucleotide molecule encoding a        modified Clostridial toxin, the polynucleotide molecule        comprising:        -   i) a polynucleotide molecule encoding a Clostridial toxin            enzymatic domain capable of executing an enzymatic target            modification step of a Clostridial toxin intoxication            process;        -   ii) a polynucleotide molecule encoding a Clostridial toxin            translocation domain capable of executing a translocation            step of a Clostridial toxin intoxication process;        -   iii) a polynucleotide molecule encoding a translocation            facilitating domain capable of facilitating a translocation            step of a Clostridial toxin intoxication process;        -   iv) a polynucleotide molecule encoding an enhanced targeting            domain comprising a Clostridial non-toxin associated protein            β-trefoil domain capable of executing a cell binding step of            a Clostridial toxin intoxication process; and        -   v) a polynucleotide molecule encoding a protease cleavage            site        -   wherein cleavage of the protease cleavage site converts the            single-chain form of the modified Clostridial toxin into the            di-chain form.    -   b) expressing the modified Clostridial toxin encoded by the        polynucleotide molecule.-   148. The method according to 147, wherein the polynucleotide    molecule is any one of the polynucleotide molecules of 118.-   149. A method of producing a modified Clostridial toxin comprising    the steps of:    -   a) introducing into a cell a polynucleotide molecule encoding a        modified Clostridial toxin, the polynucleotide molecule        comprising:        -   i) a polynucleotide molecule encoding a Clostridial toxin            enzymatic domain capable of executing an enzymatic target            modification step of a Clostridial toxin intoxication            process;        -   ii) a polynucleotide molecule encoding a Clostridial toxin            translocation domain capable of executing a translocation            step of a Clostridial toxin intoxication process;        -   iii) a polynucleotide molecule encoding a translocation            facilitating domain capable of facilitating a translocation            step of a Clostridial toxin intoxication process;        -   iv) a polynucleotide molecule encoding an enhanced targeting            domain comprising a FGF β-trefoil domain capable of            selectively binding an FGFR3; and        -   v) a polynucleotide molecule encoding a protease cleavage            site        -   wherein cleavage of the protease cleavage site converts the            single-chain form of the modified Clostridial toxin into the            di-chain form.    -   b) expressing the modified Clostridial toxin encoded by the        polynucleotide molecule.-   150. The method according to 149, wherein the polynucleotide    molecule is any one of the polynucleotide molecules of 129.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofdisclosed embodiments and are in no way intended to limit any of theembodiments disclosed in the present specification.

Example 1 Construction of a Modified Clostridial Toxin Comprising aTranslocation Facilitating Domain and a Modified Clostridial ToxinTargeting Domain with Enhanced Binding Activity

This example illustrates how to make a modified Clostridial toxincomprising a translocation facilitating domain and a modifiedClostridial toxin binding domain with enhanced binding activity usingsite-directed mutagenesis.

A polynucleotide molecule encoding BoNT/A (SEQ ID NO: 1) is synthesizedusing standard procedures (BlueHeron® Biotechnology, Bothell, Wash.).Oligonucleotides of 20 to 50 bases in length are synthesized usingstandard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/A. Thesynthesized polynucleotide molecule is verified by sequencing using BigDye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.)and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

If desired, an expression optimized polynucleotide molecule encodingBoNT/A (SEQ ID NO: 1) can be synthesized in order to improve expressionin an Escherichia coli strain. The polynucleotide molecule encoding theBoNT/A can be modified to 1) contain synonymous codons typically presentin native polynucleotide molecules of an Escherichia coli strain; 2)contain a G+C content that more closely matches the average G+C contentof native polynucleotide molecules found in an Escherichia coli strain;3) reduce polymononucleotide regions found within the polynucleotidemolecule; and/or 4) eliminate internal regulatory or structural sitesfound within the polynucleotide molecule, see, e.g., Lance E. Steward etal. Optimizing Expression of Active Botulinum Toxin Type E, PCT PatentSerial No. 2005/020578 (Jun. 9, 2005); Lance E. Steward et al.Optimizing Expression of Active Botulinum Toxin Type A, PCT PatentSerial No. 2005/027917 (Aug. 3, 2005). Once sequence optimization iscomplete, oligonucleotides of 20 to 50 bases in length are synthesizedusing standard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/A. Thesynthesized polynucleotide molecule is verified by sequencing using BigDye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.)and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.). Isso desired, optimization to a different organism, such as, e.g., a yeaststrain, an insect cell-line or a mammalian cell line, can be done, see,e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005);and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005).

A similar cloning strategy is used to make pUCBHB1 cloning constructscomprising a polynucleotide molecule encoding BoNT/B of SEQ ID NO: 2; apolynucleotide molecule encoding BoNT/C1 of SEQ ID NO: 3; apolynucleotide molecule encoding BoNT/D of SEQ ID NO: 4; apolynucleotide molecule encoding BoNT/E of SEQ ID NO: 5; apolynucleotide molecule encoding BoNT/F of SEQ ID NO: 6; apolynucleotide molecule encoding BoNT/G of SEQ ID NO: 7; and apolynucleotide molecule encoding TeNT of SEQ ID NO: 8. In addition, oneskilled in the art can modify Clostridial toxins, such as, e.g., toinclude an exogenous protease cleavage site within the di-chain loopregion, or flexible spacer regions.

To construct pET29/BoNT/A, a pUCBHB1/BoNT/A construct is digested withrestriction endonucleases that 1) excise the insert comprising the openreading frame of SEQ ID NO: 1 encoding BoNT/A; and 2) enable this insertto be operably-linked to a pET29 vector (EMD Biosciences-Novagen,Madison, Wis.). This insert is subcloned using a T4 DNA ligase procedureinto a pET29 vector that is digested with appropriate restrictionendonucleases to yield pET29/BoNT/A. The ligation mixture is transformedinto chemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 50 μg/mL of Kanamycin, and placed in a 37° C.incubator for overnight growth. Bacteria containing expressionconstructs are identified as Kanamycin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the insert. This cloningstrategy yielded a pET29 expression construct comprising thepolynucleotide molecule encoding the BoNT/A of SEQ ID NO: 1operably-linked to a carboxyl terminal polyhistidine affinity bindingpeptide.

A similar cloning strategy is used to make pET29 expression constructscomprising a polynucleotide molecule encoding BoNT/B of SEQ ID NO: 2; apolynucleotide molecule encoding BoNT/C1 of SEQ ID NO: 3; apolynucleotide molecule encoding BoNT/D of SEQ ID NO: 4; apolynucleotide molecule encoding BoNT/E of SEQ ID NO: 5; apolynucleotide molecule encoding BoNT/F of SEQ ID NO: 6; apolynucleotide molecule encoding BoNT/G of SEQ ID NO: 7; and apolynucleotide molecule encoding TeNT of SEQ ID NO: 8.

To construct a modified BoNT/A comprising a modified H_(CC) targetingdomain with enhanced binding activity, specific amino acids influencingbinding activity will be changed. It is already known that amino acidsTrp 1101, Gly 1102, Leu 1105, Tyr 1111, Tyr 1112, Gly 1158, Ile 1163,Asp 1179, Glu 1203, Phe 1252, Ser 1264, Trp 1266, Tyr 1267, Gln 1270,Gly 1279 and Trp 1282 of SEQ ID NO: 1 are important for function. Todetermine which amino acid substitutions could enhance the bindingactivity of a BoNT/A binding domain, computational protein designalgorithms will generate novel proteins with optimized properties. Thecrystal structure of a BoNT/A binding domain will be used as thestarting template for computational calculations. Potential amino acidcandidates will be identified using a combined output from ProteinDesign Automation® (PDA®) and Sequence Prediction Algorithm™ (SPA™)calculations. For PDA calculations, the conformations of amino acids atvariable positions will be represented as a set of backbone-independentside chain rotamers derived from the rotamer library. The energies ofall possible combinations of the considered amino acids at the chosenvariable positions will be calculated using a force field containingterms describing van der Waals, solvation, electrostatic, and hydrogenbond interactions. The optimal (ground state) sequence will bedetermined using a Dead End Elimination (DEE) algorithm, and a MonteCarlo (MC) algorithm will be used to evaluate the energies of similarsequences around the predicted ground state. SPA calculations utilize agenetic algorithm to screen for low energy sequences, with energiesbeing calculated during each round of “evolution” for those sequencesbeing sampled. The conformations of amino acids will be represented as aset of side chain rotamers derived from a backbone-independent rotamerlibrary using a flexible rotamer model. SPA calculations will generatesequences which will be subsequently clustered computationally intogroups of similar sequences using a nearest neighbor single linkagehierarchical clustering algorithm. Critical contact amino acids will befixed in both sequence and conformation and calculations will be carriedout to evaluate single and combinatorial substitutions at variable aminoacids. All amino acids in contact with these residues will be floated,that is the amino acid conformation but not the amino acid identity willbe allowed to vary to allow for conformational adjustments. Finalexperimental substitutions will be chosen based on their predictedenergies relative to the naturally occurring BoNT/A binding domain andtheir occupancy, that is the number times the substitution occurred inthe set of 1000 MC or genetic algorithm sequences. Two sets of designcalculations will be carried out using Rosetta to identify substitutionspredicted to stabilize the BoNT/A binding domain. In the first round,only single amino acid substitutions will be modeled. In a second round,interface amino acids will be allowed to change to all 20 naturallyoccurring amino acids including the native amino acid type, butexcluding cysteine, simultaneously. In each case, amino acid side chainscontacting the substituted amino acid side chains will be repacked(allowing all rotamers of the native amino acid type). Sequences andconformations with low energies will be selected using a Monte-Carlosimulated annealing procedure. All resulting protein complex models willbe rescored by computing a predicted binding energy. Final sequenceswere selected for the lowest binding energy

Identified candidate amino acids will be changed using site-directed invitro mutagenesis. A 50 μL reaction will be assembled using pET29/BoNT/Aas a template, sense and antisense oligonucleotides encoding the desiredamino acid change identified above, and reagents included with theQuickChange® II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla,Calif.). The polymerase chain reaction (PCR) mix will contain 5 μL of10× Buffer, 1 μL of deoxyribonucleotides (dNTPs), 1 μL of PfuUltra™ HighFidelity DNA polymerase (2.5 units/μL), 125 ng of each primer, 100 ng oftemplate DNA, and nuclease-free water to a final volume of 50 μL. Thethermocycler conditions will be: one cycle of 95° C. for 60 seconds; 16cycles of 95° C. for 30 seconds, 55° C. for 60 seconds, and 72° C. for10 minutes; one cycle of 72° C. for 5 minutes; and 4° C. to hold.Following thermocycling, 1 μL of Dpnl restriction enzyme (Stratagene, LaJolla, Calif.) will be added to the reaction and will be incubated for 1hour at 37° C. to digest the template DNA. The reaction will be purifiedby QIAquick kit (QIAGEN, Inc., Valencia, Calif.) and will be analysis byagarose gel electrophoresis to determine that the reaction producedfull-length plasmid. The mutagenesis products will be transformed intochemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 100 μg/mL of Ampicillin, and will be placedin a 37° C. incubator for overnight growth. Candidate mutagenesisconstructs will be isolated as Ampicillin resistant colonies and will beanalyzed using an alkaline lysis plasmid mini-preparation procedure toisolate the expression construct and restriction endonuclease digests todetermine the presence of the insert. The incorporation of the pointmutation will be determined by sequence analysis of candidate plasmidconstructs.

To test the binding activity of modified BoNT/A candidates with enhancedbinding activity, the soluble portion of FGFR3 will be expressedrecombinantly for use in surface plasmon resonance (SPR) binding assays,e.g., Biacore® (Biacore Inc., Piscataway, N.J.). The soluble portion ofFGFR3 will be expressed as a fusion to streptavidin and the receptorwill then be immobilized on an appropriate sensor chip. Utilizing aBiacore® instrument, changes in local refractive index as a result ofreceptor binding will be measured as a change in the SPR angle. Therates of change in the SPR angle will then be analyzed to determineassociation rate (K_(on)), dissociation rate (K_(off)) and thedissociation equilibrium constant (K_(D)=K_(off)/K_(on)). ModifiedBoNT/A candidates with enhanced binding activity will exhibit either anincreased association rate, a decreased dissociation rate, both anincreased association rate and a decreased dissociation rate, or adecreased dissociation equilibrium constant relative to the measurementsobtained from the naturally occurring BoNT/A from which the modifiedBoNT/A with enhanced binding activity was derived.

To construct a modified BoNT/A described above that replaces the BoNT/Atranslocation facilitating domain with another Clostridial toxintranslocation facilitating domain, a translocation facilitating domainof BoNT/B will be introduced into the modified BoNT/A described aboveusing a Splicing by Overlapping ends polymerase chain reaction (SOE-PCR)procedure, see, e.g., R. M. Horton et al., Engineering hybrid geneswithout the use of restriction enzymes: gene splicing by overlappingextension, 77(1) Gene 61-68 (1989); and R. M. Horton, PCR-mediatedrecombination and mutagenesis. SOEing together tailor-made genes, 3(2)Mol. Biotechnol. 93-99 (1995). A nucleic acid fragment comprising aregion encoding amino acids 859 to 1097 of BoNT/B (SEQ ID NO: 2) will beoperably-linked by SOE-PCR to replace the region corresponding to theBoNT/A translocation facilitating domain of amino acids 874-1110 of SEQID NO: 1 of the modified BoNT/A described above and will be subclonedinto a pCR2.1 vector using the TOPO® TA cloning method (Invitrogen, Inc,Carlsbad, Calif.). The forward and reverse oligonucleotide primers usedfor these reactions are designed to include unique restriction enzymesites useful for subsequent subcloning steps. The resulting construct isdigested with restriction enzymes that 1) excise the insert containingthe entire open reading frame encoding the modified BoNT/A; and 2)enable this insert to be operably-linked to a pQBI-25A vector (Qbiogene,Inc., Carlsbad, Calif.). The resulting restriction fragment will bepurified by the QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia,Calif.), and will be subcloned using a T4 DNA ligase procedure into apQBI-25A vector (Qbiogene, Inc., Irvine, Calif.). This cloning strategyyielded a pQBI-25 expression construct encoding a modified BoNT/Acomprising a BoNT/B translocation facilitating domain and a modifiedH_(CC) targeting domain with enhanced binding activity.

A similar cloning strategy is used to make pQBI-25 expression constructscomprising a polynucleotide molecule encoding a modified BoNT/Acomprising a modified targeting domain with enhanced binding activitythat includes, e.g., a translocation facilitating domain comprisingamino acids 869-1111 of BoNT/C1 of SEQ ID NO: 3; a translocationfacilitating domain comprising amino acids 865-1098 of BoNT/D of SEQ IDNO: 4; a translocation facilitating domain comprising amino acids846-1058 of BoNT/E of SEQ ID NO: 5; a translocation facilitating domaincomprising amino acids 867-1105 of BoNT/F of SEQ ID NO: 6; atranslocation facilitating domain comprising amino acids 866-1105 ofBoNT/G of SEQ ID NO: 7; or a translocation facilitating domaincomprising amino acids 882-1127 of TeNT of SEQ ID NO: 8.

Example 2 Construction of a Modified Clostridial Toxin Comprising aTranslocation Facilitating Domain and a Non-Toxin Associated Protein

The following example illustrates how to make a modified Clostridialtoxin comprising a translocation facilitating domain and an enhancedtargeting domain comprising a non-toxin associated protein.

A polynucleotide molecule encoding BoNT/A-33/A is synthesized usingstandard procedures (BlueHeron® Biotechnology, Bothell, Wash.), asdescribed in Example 1. BoNT/A-33/A is a BoNT/A modified to replaceamino acids 1111-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, withamino acids 151 to 293 of SEQ ID NO: 9, a HA-33 β-trefoil domain from aClostridial botulinum serotype A strain. If desired, an expressionoptimized polynucleotide molecule encoding BoNT/A-33/A can besynthesized in order to improve expression in to a different organism,such as, e.g., an Escherichia coli strain, a yeast strain, an insectcell-line or a mammalian cell line, can be done, see, e.g., Steward,supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); and Steward,supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). The synthesizedpolynucleotide molecule is verified by sequencing using Big DyeTerminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.) andan ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructsfor BoNT/B-33/A, a modified BoNT/B where amino acids 1098-1291 of SEQ IDNO: 2 are replaced with amino acids 151 to 293 of SEQ ID NO: 9;BoNT/C1-33/A, a modified BoNT/C1 where amino acids 1112-1291 of SEQ IDNO: 3 are replaced with amino acids 151 to 293 of SEQ ID NO: 9;BoNT/D-33/A, a modified BoNT/D where amino acids 1099-1276 of SEQ ID NO:4 are replaced with amino acids 151 to 293 of SEQ ID NO: 9; BoNT/E-33/A,a modified BoNT/E where amino acids 1086-1252 of SEQ ID NO: 5 arereplaced with amino acids 151 to 293 of SEQ ID NO: 9; BoNT/F-33/A, amodified BoNT/F where amino acids 1106-1274 of SEQ ID NO: 6 are replacedwith amino acids 151 to 293 of SEQ ID NO: 9; BoNT/G-33/A, a modifiedBoNT/G where amino acids 1106-1297 of SEQ ID NO: 7 are replaced withamino acids 151 to 293 of SEQ ID NO: 9; and TeNT-33/A, a modified TeNTwhere amino acids 1128-1315 of SEQ ID NO: 8 are replaced with aminoacids 151 to 293 of SEQ ID NO: 9. Similarly, the β-trefoil domain from aClostridial toxin indicated above can be replaced with a non-toxinassociated protein β-trefoil domain comprising amino acids 10-144 of SEQID NO: 9; amino acids 10-144 of SEQ ID NO: 10; amino acids 10-144 of SEQID NO: 11; amino acids 10-146 of SEQ ID NO: 12; amino acids 10-144 ofSEQ ID NO: 13; amino acids 10-144 of SEQ ID NO: 14; amino acids 10-146of SEQ ID NO: 15; amino acids 10-141 of SEQ ID NO: 16; amino acids10-141 of SEQ ID NO: 17; amino acids 10-141 of SEQ ID NO: 18; aminoacids 151-293 of SEQ ID NO: 10; amino acids 151-293 of SEQ ID NO: 11;amino acids 153-294 of SEQ ID NO: 12; amino acids 151-279 of SEQ ID NO:13; amino acids 151-292 of SEQ ID NO: 14; amino acids 153-291 of SEQ IDNO: 15; amino acids 148-285 of SEQ ID NO: 16; amino acids 148-286 of SEQID NO: 17; amino acids 148-286 of SEQ ID NO: 18; amino acids 9-146 ofSEQ ID NO: 19; amino acids 9-146 of SEQ ID NO: 20; amino acids 9-146 ofSEQ ID NO: 21; amino acids 9-146 of SEQ ID NO: 22; amino acids 1050-1194of SEQ ID NO: 23; amino acids 1050-1199 of SEQ ID NO: 24; amino acids1050-1194 of SEQ ID NO: 25; amino acids 1049-1198 of SEQ ID NO: 26;amino acids 1049-1197 of SEQ ID NO: 27; amino acids 1049-1197 of SEQ IDNO: 28; amino acids 1014-1163 of SEQ ID NO: 29; amino acids 1016-1160 ofSEQ ID NO: 30; amino acids 1017-1166 of SEQ ID NO: 31; and amino acids1050-1199 of SEQ ID NO: 32.

To construct pET29/BoNT/A-33/A, a pUCBHB1/BoNT/A-33/A construct isdigested with restriction endonucleases that 1) excise the insertcomprising the open reading frame encoding BoNT/A-33/A; and 2) enablethis insert to be operably-linked to a pET29 vector (EMDBiosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4DNA ligase procedure into a pET29 vector that is digested withappropriate restriction endonucleases to yield pET29/BoNT/A-33/A. Theligation mixture is transformed into chemically competent E. coli DH5αcells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method,plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL ofKanamycin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Kanamycinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe insert. This cloning strategy yielded a pET29 expression constructcomprising the polynucleotide molecule encoding BoNT/A-33/Aoperably-linked to a carboxyl terminal polyhistidine affinity bindingpeptide.

A similar cloning strategy is used to make pET29 expression constructscomprising a polynucleotide molecule encoding for BoNT/B-33/A,BoNT/C1-33/A, BoNT/D-33/A, BoNT/E-33/A, BoNT/F-33/A, BoNT/G-33/A,TeNT-33/A, as well as the modified Clostridial toxin indicated abovecomprising amino acids 10-144 of SEQ ID NO: 9; amino acids 10-144 of SEQID NO: 10; amino acids 10-144 of SEQ ID NO: 11; amino acids 10-146 ofSEQ ID NO: 12; amino acids 10-144 of SEQ ID NO: 13; amino acids 10-144of SEQ ID NO: 14; amino acids 10-146 of SEQ ID NO: 15; amino acids10-141 of SEQ ID NO: 16; amino acids 10-141 of SEQ ID NO: 17; aminoacids 10-141 of SEQ ID NO: 18; amino acids 151-293 of SEQ ID NO: 10;amino acids 151-293 of SEQ ID NO: 11; amino acids 153-294 of SEQ ID NO:12; amino acids 151-279 of SEQ ID NO: 13; amino acids 151-292 of SEQ IDNO: 14; amino acids 153-291 of SEQ ID NO: 15; amino acids 148-285 of SEQID NO: 16; amino acids 148-286 of SEQ ID NO: 17; amino acids 148-286 ofSEQ ID NO: 18; amino acids 9-146 of SEQ ID NO: 19; amino acids 9-146 ofSEQ ID NO: 20; amino acids 9-146 of SEQ ID NO: 21; amino acids 9-146 ofSEQ ID NO: 22; amino acids 1050-1194 of SEQ ID NO: 23; amino acids1050-1199 of SEQ ID NO: 24; amino acids 1050-1194 of SEQ ID NO: 25;amino acids 1049-1198 of SEQ ID NO: 26; amino acids 1049-1197 of SEQ IDNO: 27; amino acids 1049-1197 of SEQ ID NO: 28; amino acids 1014-1163 ofSEQ ID NO: 29; amino acids 1016-1160 of SEQ ID NO: 30; amino acids1017-1166 of SEQ ID NO: 31; and amino acids 1050-1199 of SEQ ID NO: 32.

To construct a BoNT/A-33/A that replaces the BoNT/A translocationfacilitating domain with another Clostridial toxin translocationfacilitating domain, a translocation facilitating domain of BoNT/B willbe introduced into the BoNT/A-33/A as described above using a Splicingby Overlapping ends polymerase chain reaction (SOE-PCR) procedure, see,e.g., R. M. Horton et al., Engineering hybrid genes without the use ofrestriction enzymes: gene splicing by overlapping extension, 77(1) Gene61-68 (1989); and R. M. Horton, PCR-mediated recombination andmutagenesis. SOEing together tailor-made genes, 3(2) Mol. Biotechnol.93-99 (1995). A nucleic acid fragment comprising a region encoding aminoacids 859 to 1097 of BoNT/B (SEQ ID NO: 2) will be operably-linked bySOE-PCR to replace the region corresponding to the BoNT/A translocationfacilitating domain comprising amino acids 874-1110 of SEQ ID NO: 1 ofthe BoNT/A-33/A and will be subcloned into a pCR2.1 vector using theTOPO® TA cloning method (Invitrogen, Inc, Carlsbad, Calif.). The forwardand reverse oligonucleotide primers used for these reactions aredesigned to include unique restriction enzyme sites useful forsubsequent subcloning steps. The resulting construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding the modified BoNT/A-33/A; and 2) enable thisinsert to be operably-linked to a pQBI-25A vector (Qbiogene, Inc.,Carlsbad, Calif.). The resulting restriction fragment will be purifiedby the QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, Calif.), andwill be subcloned using a T4 DNA ligase procedure into a pQBI-25A vector(Qbiogene, Inc., Irvine, Calif.). This cloning strategy yielded apQBI-25 expression construct encoding a BoNT/A-33/A comprising a BoNT/Btranslocation facilitating domain.

A similar cloning strategy is used to make pQBI-25 expression constructscomprising a polynucleotide molecule encoding a BoNT/A-33/A thatincludes, e.g., a translocation facilitating domain comprising aminoacids 869-1111 of BoNT/C1 of SEQ ID NO: 3; a translocation facilitatingdomain comprising amino acids 865-1098 of BoNT/D of SEQ ID NO: 4; atranslocation facilitating domain comprising amino acids 846-1058 ofBoNT/E of SEQ ID NO: 5; a translocation facilitating domain comprisingamino acids 867-1105 of BoNT/F of SEQ ID NO: 6; a translocationfacilitating domain comprising amino acids 866-1105 of BoNT/G of SEQ IDNO: 7; or a translocation facilitating domain comprising amino acids882-1127 of TeNT of SEQ ID NO: 8, as well as the modified Clostridialtoxin indicated above comprising amino acids 10-144 of SEQ ID NO: 9;amino acids 10-144 of SEQ ID NO: 10; amino acids 10-144 of SEQ ID NO:11; amino acids 10-146 of SEQ ID NO: 12; amino acids 10-144 of SEQ IDNO: 13; amino acids 10-144 of SEQ ID NO: 14; amino acids 10-146 of SEQID NO: 15; amino acids 10-141 of SEQ ID NO: 16; amino acids 10-141 ofSEQ ID NO: 17; amino acids 10-141 of SEQ ID NO: 18; amino acids 151-293of SEQ ID NO: 10; amino acids 151-293 of SEQ ID NO: 11; amino acids153-294 of SEQ ID NO: 12; amino acids 151-279 of SEQ ID NO: 13; aminoacids 151-292 of SEQ ID NO: 14; amino acids 153-291 of SEQ ID NO: 15;amino acids 148-285 of SEQ ID NO: 16; amino acids 148-286 of SEQ ID NO:17; amino acids 148-286 of SEQ ID NO: 18; amino acids 9-146 of SEQ IDNO: 19; amino acids 9-146 of SEQ ID NO: 20; amino acids 9-146 of SEQ IDNO: 21; amino acids 9-146 of SEQ ID NO: 22; amino acids 1050-1194 of SEQID NO: 23; amino acids 1050-1199 of SEQ ID NO: 24; amino acids 1050-1194of SEQ ID NO: 25; amino acids 1049-1198 of SEQ ID NO: 26; amino acids1049-1197 of SEQ ID NO: 27; amino acids 1049-1197 of SEQ ID NO: 28;amino acids 1014-1163 of SEQ ID NO: 29; amino acids 1016-1160 of SEQ IDNO: 30; amino acids 1017-1166 of SEQ ID NO: 31; and amino acids1050-1199 of SEQ ID NO: 32.

Example 3 Construction of a Modified Clostridial Toxin Comprising aTranslocation Facilitating Domain and a Non-Toxin Associated ProteinFold

The following example illustrates how to make a modified Clostridialtoxin comprising a translocation facilitating domain and an enhancedtargeting domain comprising a non-toxin associated protein fold.

A polynucleotide molecule encoding BoNT/A-17γ/A is synthesized usingstandard procedures (BlueHeron® Biotechnology, Bothell, Wash.), asdescribed in Example 1. BoNT/A-17γ/A is a BoNT/A modified to replaceamino acids 1237-1296 of SEQ ID NO: 1, a γ-fold from a BoNT/A β-trefoildomain, with amino acids 95 to 146 of SEQ ID NO: 19, a γ-fold from aHA-17 β-trefoil domain from a Clostridial botulinum serotype A strain.If desired, an expression optimized polynucleotide molecule encodingBoNT/A-17γ/A can be synthesized in order to improve expression in to adifferent organism, such as, e.g., an Escherichia coli strain, a yeaststrain, an insect cell-line or a mammalian cell line, can be done, see,e.g., Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005);and Steward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005).The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructsfor BoNT/B-17γ/A, a modified BoNT/B where amino acids 1223-1291 of SEQID NO: 2 are replaced with amino acids 95 to 146 of SEQ ID NO: 19;BoNT/C1-F18γ, a modified BoNT/C1 where amino acids 1230-1291 of SEQ IDNO: 3 are replaced with amino acids 95 to 146 of SEQ ID NO: 19;BoNT/D-17γ/A, a modified BoNT/D where amino acids 1219-1276 of SEQ IDNO: 4 are replaced with amino acids 95 to 146 of SEQ ID NO: 19;BoNT/E-17γ/A, a modified BoNT/E where amino acids 1199-1252 of SEQ IDNO: 5 are replaced with amino acids 95 to 146 of SEQ ID NO: 19;BoNT/F-17γ/A, a modified BoNT/F where amino acids 1222-1274 of SEQ IDNO: 6 are replaced with amino acids 95 to 146 of SEQ ID NO: 19;BoNT/G-17γ/A, a modified BoNT/G where amino acids 1231-1297 of SEQ IDNO: 7 are replaced with amino acids 95 to 146 of SEQ ID NO: 19; andTeNT-17γ/A, a modified TeNT where amino acids 1255-1315 of SEQ ID NO: 8are replaced with amino acids 95 to 146 of SEQ ID NO: 19.

Similarly, the γ-fold from a HA-17/A β-trefoil domain can replace aminoacids 1111-1162 of SEQ ID NO: 1, an α-fold from a BoNT/A β-trefoildomain; amino acids 1098-1147 of SEQ ID NO: 2, an α-fold from a BoNT/Bβ-trefoil domain; amino acids 1112-1150 of SEQ ID NO: 3, an α-fold froma BoNT/C1 trefoil domain; amino acids 1099-1137 of SEQ ID NO: 4, anα-fold from a BoNT/D β-trefoil domain; amino acids 1086-1129 of SEQ IDNO: 5, an α-fold from a BoNT/E β-trefoil domain; amino acids 1106-1152of SEQ ID NO: 6, an α-fold from a BoNT/F β-trefoil domain; amino acids1106-1153 of SEQ ID NO: 7, an α-fold from a BoNT/G β-trefoil domain; oramino acids 1128-1177 of SEQ ID NO: 8, an α-fold from a TeNT β-trefoildomain. Similarly, the γ-fold from a HA-17/Aβ-trefoil domain can replaceamino acids 1179-1223 of SEQ ID NO: 1, a β-fold from a BoNT/A β-trefoildomain; amino acids 1166-1210 of SEQ ID NO: 2, a β-fold from a BoNT/Bβ-trefoil domain; amino acids 1167-1218 of SEQ ID NO: 3, a β-fold from aBoNT/C1 β-trefoil domain; amino acids 1154-1207 of SEQ ID NO: 4, aβ-fold from a BoNT/D β-trefoil domain; amino acids 1147-1190 of SEQ IDNO: 5, a β-fold from a BoNT/E β-trefoil domain; amino acids 1172-1213 ofSEQ ID NO: 6, a β-fold from a BoNT/F β-trefoil domain; amino acids1173-1218 of SEQ ID NO: 7, a β-fold from a BoNT/G β-trefoil domain; oramino acids 1195-1240 of SEQ ID NO: 8, a β-fold from a TeNT β-trefoildomain.

Similarly, an α-fold, β-fold or γ-fold from a β-trefoil domain from aClostridial toxin indicated above can be replaced with a HA-17 γ-foldcomprising amino acids 95-146 of SEQ ID NO: 20, amino acids 95-146 ofSEQ ID NO: 21, or amino acids 95-146 of SEQ ID NO: 22; a HA-33 γ-foldcomprising amino acids 105-144 of SEQ ID NO: 9, amino acids 105-144 ofSEQ ID NO: 10, amino acids 105-144 of SEQ ID NO: 11, amino acids 107-146of SEQ ID NO: 12, amino acids 105-144 of SEQ ID NO: 13, amino acids105-144 of SEQ ID NO: 14, amino acids 107-146 of SEQ ID NO: 15, aminoacids 103-141 of SEQ ID NO: 16, amino acids 103-141 of SEQ ID NO: 17,amino acids 103-141 of SEQ ID NO: 18, amino acids 249-293 of SEQ ID NO:9, amino acids 249-293 of SEQ ID NO: 10, amino acids 249-293 of SEQ IDNO: 11, amino acids 250-294 of SEQ ID NO: 12, amino acids 249-279 of SEQID NO: 13, amino acids 248-292 of SEQ ID NO: 14, amino acids 249-291 ofSEQ ID NO: 15, amino acids 241-285 of SEQ ID NO: 16, amino acids 242-286of SEQ ID NO: 17, or amino acids 242-286 of SEQ ID NO: 18; or a NTNHγ-fold comprising amino acids 1149-1194 of SEQ ID NO: 23, amino acids1149-1199 of SEQ ID NO: 24, amino acids 1149-1194 of SEQ ID NO: 25,amino acids 1148-1198 of SEQ ID NO: 26, amino acids 1148-1197 of SEQ IDNO: 27, amino acids 1148-1197 of SEQ ID NO: 28, amino acids 1114-1163 ofSEQ ID NO: 29, amino acids 1115-1160 of SEQ ID NO: 30, amino acids1117-1166 of SEQ ID NO: 31, or amino acids 1150-1199 of SEQ ID NO: 32.

Likewise, an α-fold, β-fold or γ-fold from a β-trefoil domain from aClostridial toxin indicated above can be replaced with a HA-17 α-foldcomprising amino acids 9-50 of SEQ ID NO: 19, amino acids 9-50 of SEQ IDNO: 20, amino acids 9-50 of SEQ ID NO: 21, or amino acids 9-50 of SEQ IDNO: 22; a HA-33 α-fold comprising amino acids 10-54 of SEQ ID NO: 9,amino acids 10-54 of SEQ ID NO: 10, amino acids 10-54 of SEQ ID NO: 11,amino acids 10-56 of SEQ ID NO: 12, amino acids 10-54 of SEQ ID NO: 13,amino acids 10-54 of SEQ ID NO: 14, amino acids 10-56 of SEQ ID NO: 15,amino acids 10-54 of SEQ ID NO: 16, amino acids 10-54 of SEQ ID NO: 17,amino acids 10-54 of SEQ ID NO: 18, amino acids 151-195 of SEQ ID NO: 9,amino acids 151-195 of SEQ ID NO: 10, amino acids 151-195 of SEQ ID NO:11, amino acids 153-197 of SEQ ID NO: 12, amino acids 151-195 of SEQ IDNO: 13, amino acids 151-195 of SEQ ID NO: 14, amino acids 153-197 of SEQID NO: 15, amino acids 148-190 of SEQ ID NO: 16, amino acids 148-190 ofSEQ ID NO: 17, or amino acids 148-190 of SEQ ID NO: 18; or a NTNH α-foldcomprising amino acids 1050-1097 of SEQ ID NO: 23, amino acids 1050-1097of SEQ ID NO: 24, amino acids 1050-1097 of SEQ ID NO: 25, amino acids1049-1096 of SEQ ID NO: 26, amino acids 1049-1096 of SEQ ID NO: 27,amino acids 1049-1096 of SEQ ID NO: 28, amino acids 1014-1061 of SEQ IDNO: 29, amino acids 1016-1063 of SEQ ID NO: 30, amino acids 1017-1064 ofSEQ ID NO: 31, or amino acids 1050-1097 of SEQ ID NO: 32.

Further, an α-fold, β-fold or γ-fold from a β-trefoil domain from aClostridial toxin indicated above can be replaced with a HA-17 β-foldcomprising amino acids 55-91 of SEQ ID NO: 19, amino acids 55-91 of SEQID NO: 20, amino acids 55-91 of SEQ ID NO: 21, or amino acids 55-91 ofSEQ ID NO: 22; a HA-33 β-fold comprising amino acids 60-100 of SEQ IDNO: 9, amino acids 60-100 of SEQ ID NO: 10, amino acids 60-100 of SEQ IDNO: 11, amino acids 62-102 of SEQ ID NO: 12, amino acids 60-100 of SEQID NO: 13, amino acids 60-100 of SEQ ID NO: 14, amino acids 62-102 ofSEQ ID NO: 15, amino acids 60-98 of SEQ ID NO: 16, amino acids 60-98 ofSEQ ID NO: 17, amino acids 60-98 of SEQ ID NO: 18, amino acids 200-242of SEQ ID NO: 9, amino acids 200-242 of SEQ ID NO: 10, amino acids200-242 of SEQ ID NO: 11, amino acids 202-243 of SEQ ID NO: 12, aminoacids 200-242 of SEQ ID NO: 13, amino acids 200-241 of SEQ ID NO: 14,amino acids 200-242 of SEQ ID NO: 15, amino acids 195-234 of SEQ ID NO:16, amino acids 195-235 of SEQ ID NO: 17, or amino acids 195-235 of SEQID NO: 18; or a NTNH β-fold comprising amino acids 1111-1138 of SEQ IDNO: 23, amino acids 1111-1139 of SEQ ID NO: 24, amino acids 1111-1138 ofSEQ ID NO: 25, amino acids 1110-1138 of SEQ ID NO: 26, amino acids1110-1138 of SEQ ID NO: 27, amino acids 1110-1138 of SEQ ID NO: 28,amino acids 1075-1103 of SEQ ID NO: 29, amino acids 1077-1104 of SEQ IDNO: 30, amino acids 1078-1106 of SEQ ID NO: 31, or amino acids 1111-1139of SEQ ID NO: 32.

To construct pET29/BoNT/A-17γ/A, a pUCBHB1/BoNT/A-17γ/A construct isdigested with restriction endonucleases that 1) excise the insertcomprising the open reading frame encoding BoNT/A-17γ/A; and 2) enablethis insert to be operably-linked to a pET29 vector (EMDBiosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4DNA ligase procedure into a pET29 vector that is digested withappropriate restriction endonucleases to yield pET29/BoNT/A-17γ/A. Theligation mixture is transformed into chemically competent E. coli DH5αcells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method,plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL ofKanamycin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Kanamycinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe insert. This cloning strategy yielded a pET29 expression constructcomprising the polynucleotide molecule encoding BoNT/A-17γ/Aoperably-linked to a carboxyl terminal polyhistidine affinity bindingpeptide.

A similar cloning strategy is used to make pET29 expression constructscomprising a polynucleotide molecule encoding BoNT/B-17γ/A,BoNT/C1-17γ/A, BoNT/D-17γ/A, BoNT/E-17γ/A, BoNT/F-17γ/A, BoNT/G-17γ/A,TeNT-17γ/A, BoNT/A-33-1γ/A, BoNT/B-33-1γ/A, BoNT/C1-33-1γ/A,BoNT/D-33-1γ/A, BoNT/E-33-1γ/A, BoNT/F-33-1γ/A, BoNT/G-33-1γ/A,TeNT-33-1γ/A, BoNT/A-33-2γ/A, BoNT/B-33-2γ/A, BoNT/C1-33-2γ/A,BoNT/D-33-2γ/A, BoNT/E-33-2γ/A, BoNT/F-33-2γ/A, BoNT/G-33-2γ/A,TeNT-33-2γ/A, BoNT/B-NTNHγ/A, BoNT/B-NTNHγ/A, BoNT/C1-NTNHγ/A,BoNT/D-NTNHγ/A, BoNT/E-NTNHγ/A, BoNT/F-NTNHγ/A, BoNT/G-NTNHγ/A,TeNT-NTNHγ/A, BoNT/A-17α/A, BoNT/B-17α/A, BoNT/C1-17α/A, BoNT/D-17α/A,BoNT/E-17α/A, BoNT/F-17α/A, BoNT/G-17α/A, TeNT-17α/A, BoNT/A-33-1α/A,BoNT/B-33-1α/A, BoNT/C1-33-1α/A, BoNT/D-33-1α/A, BoNT/E-33-1α/A,BoNT/F-33-1α/A, BoNT/G-33-1α/A, TeNT-33-1α/A, BoNT/A-33-2α/A,BoNT/B-33-2α/A, BoNT/C1-33-2α/A, BoNT/D-33-2α/A, BoNT/E-33-2α/A,BoNT/F-33-2α/A, BoNT/G-33-2α/A, TeNT-33-2α/A, BoNT/B-NTNHα/A,BoNT/B-NTNHα/A, BoNT/C1-NTNHα/A, BoNT/D-NTNHα/A, BoNT/E-NTNHα/A,BoNT/F-NTNHα/A, BoNT/G-NTNHα/A, TeNT-NTNHα, BoNT/A-17β/A, BoNT/B-17β/A,BoNT/C1-17β/A, BoNT/D-17β/A, BoNT/E-17β/A, BoNT/F-17β/A, BoNT/G-17β/A,TeNT-17β/A, BoNT/A-33-1β/A, BoNT/B-33-1β/A, BoNT/C1-33-1β/A,BoNT/D-33-1β/A, BoNT/E-33-1β/A, BoNT/F-33-1β/A, BoNT/G-33-1β/A,TeNT-33-1β/A, BoNT/A-33-2β/A, BoNT/B-33-2β/A, BoNT/C1-33-2β/A,BoNT/D-33-2β/A, BoNT/E-33-2β/A, BoNT/F-33-213/A, BoNT/G-33-2β/A,TeNT-33-2β/A, BoNT/B-NTNHβ/A, BoNT/B-NTNHβ/A, BoNT/C1-NTNHβ/A,BoNT/D-NTNHβ/A, BoNT/E-NTNHWA, BoNT/F-NTNHβ/A, BoNT/G-NTNHWA,TeNT-NTNHβ.

A similar cloning strategy is also used to make pET29 expressionconstructs comprising a polynucleotide molecule encoding a modifiedClostridial toxin indicated above that has its α-fold or β-fold replacedby amino acids comprising a HA-17 α-fold, β-fold or γ-fold; amino acidscomprising a HA-33 1α-fold, 1β-fold, 1γ-fold, 2α-fold, 2β-fold, or2γ-fold; or amino acids comprising a NTNH α-fold, β-fold or γ-fold.

To construct a BoNT/A-17γ/A that replaces the BoNT/A translocationfacilitating domain with another Clostridial toxin translocationfacilitating domain, a translocation facilitating domain of BoNT/B willbe introduced into the BoNT/A-17γ/A as described above using a Splicingby Overlapping ends polymerase chain reaction (SOE-PCR) procedure, see,e.g., R. M. Horton et al., Engineering hybrid genes without the use ofrestriction enzymes: gene splicing by overlapping extension, 77(1) Gene61-68 (1989); and R. M. Horton, PCR-mediated recombination andmutagenesis. SOEing together tailor-made genes, 3(2) Mol. Biotechnol.93-99 (1995). A nucleic acid fragment comprising a region encoding aminoacids 859 to 1097 of BoNT/B (SEQ ID NO: 2) will be operably-linked bySOE-PCR to replace the region corresponding to the BoNT/A translocationfacilitating domain comprising amino acids 874-1110 of SEQ ID NO: 1 ofthe BoNT/A-17γ/A and will be subcloned into a pCR2.1 vector using theTOPO® TA cloning method (Invitrogen, Inc, Carlsbad, Calif.). The forwardand reverse oligonucleotide primers used for these reactions aredesigned to include unique restriction enzyme sites useful forsubsequent subcloning steps. The resulting construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding the modified BoNT/A-17γ/A; and 2) enable thisinsert to be operably-linked to a pQBI-25A vector (Qbiogene, Inc.,Carlsbad, Calif.). The resulting restriction fragment will be purifiedby the QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, Calif.), andwill be subcloned using a T4 DNA ligase procedure into a pQBI-25A vector(Qbiogene, Inc., Irvine, Calif.). This cloning strategy yielded apQBI-25 expression construct encoding a BoNT/A-17γ/A comprising a BoNT/Btranslocation facilitating domain.

A similar cloning strategy is used to make pQBI-25 expression constructscomprising a polynucleotide molecule encoding a BoNT/A-17γ/A thatincludes, e.g., a translocation facilitating domain comprising aminoacids 869-1111 of BoNT/C1 of SEQ ID NO: 3; a translocation facilitatingdomain comprising amino acids 865-1098 of BoNT/D of SEQ ID NO: 4; atranslocation facilitating domain comprising amino acids 846-1058 ofBoNT/E of SEQ ID NO: 5; a translocation facilitating domain comprisingamino acids 867-1105 of BoNT/F of SEQ ID NO: 6; a translocationfacilitating domain comprising amino acids 866-1105 of BoNT/G of SEQ IDNO: 7; or a translocation facilitating domain comprising amino acids882-1127 of TeNT of SEQ ID NO: 8.

Likewise, as well as, a polynucleotide molecule encoding a Clostridialtranslocation facilitating domain as described above can be introducedinto a polynucleotide molecule encoding BoNT/B-17γ/A, BoNT/C1-17γ/A,BoNT/D-17γ/A, BoNT/E-17γ/A, BoNT/F-17γ/A, BoNT/G-17γ/A, TeNT-17γ/A,BoNT/A-33-1γ/A, BoNT/B-33-1γ/A, BoNT/C1-33-1γ/A, BoNT/D-33-1γ/A,BoNT/E-33-1γ/A, BoNT/F-33-1γ/A, BoNT/G-33-1γ/A, TeNT-33-1γ/A,BoNT/A-33-2γ/A, BoNT/B-33-2γ/A, BoNT/C1-33-2γ/A, BoNT/D-33-2γ/A,BoNT/E-33-2γ/A, BoNT/F-33-2γ/A, BoNT/G-33-2γ/A, TeNT-33-2γ/A,BoNT/B-NTNHγ/A, BoNT/B-NTNHγ/A, BoNT/C1-NTNHγ/A, BoNT/D-NTNHγ/A,BoNT/E-NTNHγ/A, BoNT/F-NTNHγ/A, BoNT/G-NTNHγ/A, TeNT-NTNHγ/A,BoNT/A-17α/A, BoNT/B-17α/A, BoNT/C1-17α/A, BoNT/D-17α/A, BoNT/E-17α/A,BoNT/F-17α/A, BoNT/G-17α/A, TeNT-17α/A, BoNT/A-33-1α/A, BoNT/B-33-1α/A,BoNT/C1-33-1α/A, BoNT/D-33-1α/A, BoNT/E-33-1α/A, BoNT/F-33-1α/A,BoNT/G-33-1α/A, TeNT-33-1α/A, BoNT/A-33-2α/A, BoNT/B-33-2α/A,BoNT/C1-33-2α/A, BoNT/D-33-2α/A, BoNT/E-33-2α/A, BoNT/F-33-2α/A,BoNT/G-33-2α/A, TeNT-33-2α/A, BoNT/B-NTNHα/A, BoNT/B-NTNHα/A,BoNT/C1-NTNHα/A, BoNT/D-NTNHα/A, BoNT/E-NTNHα/A, BoNT/F-NTNHα/A,BoNT/G-NTNHα/A, TeNT-NTNHα, BoNT/A-17β/A, BoNT/B-17β/A, BoNT/C1-17β/A,BoNT/D-17β/A, BoNT/E-17β/A, BoNT/F-17β/A, BoNT/G-17β/A, TeNT-17β/A,BoNT/A-33-1β/A, BoNT/B-33-1β/A, BoNT/C1-33-1β/A, BoNT/D-33-1β/A,BoNT/E-33-1β/A, BoNT/F-33-1β/A, BoNT/G-33-1β/A, TeNT-33-1β/A,BoNT/A-33-2β/A, BoNT/B-33-2β/A, BoNT/C1-33-2β/A, BoNT/D-33-2β/A,BoNT/E-33-2β/A, BoNT/F-33-2β/A, BoNT/G-33-2β/A, TeNT-33-2β/A,BoNT/B-NTNHβ/A, BoNT/B-NTNHβ/A, BoNT/C1-NTNHβ/A, BoNT/D-NTNHβ/A,BoNT/E-NTNHβ/A, BoNT/F-NTNHβ/A, BoNT/G-NTNHβ/A, TeNT-NTNHβ.

Example 4 Construction of a Modified Clostridial Toxin Comprising aTranslocation Facilitating Domain and a FGF that Selectively Binds to aFGFR3

The following example illustrates how to make a modified Clostridialtoxin comprising a translocation facilitating domain and an enhancedtargeting domain comprising a FGF that selectively binds to a FGFR3.

A polynucleotide molecule encoding BoNT/A-F18 is synthesized usingstandard procedures (BlueHeron® Biotechnology, Bothell, Wash.), asdescribed in Example 1. BoNT/A-F18 is a BoNT/A modified to replace aminoacids 1111-1296 of SEQ ID NO: 1, a BoNT/A β-trefoil domain, with aminoacids 54 to 183 of SEQ ID NO: 39, a FGF-18 β-trefoil domain. If desired,an expression optimized polynucleotide molecule encoding BoNT/A-F18 canbe synthesized in order to improve expression in to a differentorganism, such as, e.g., an Escherichia coli strain, a yeast strain, aninsect cell-line or a mammalian cell line, can be done, see, e.g.,Steward, supra, PCT Patent Serial No. 2005/020578 (Jun. 9, 2005); andSteward, supra, PCT Patent Serial No. 2005/027917 (Aug. 3, 2005). Thesynthesized polynucleotide molecule is verified by sequencing using BigDye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.)and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructsfor BoNT/B-F18, a modified BoNT/B where amino acids 1098-1291 of SEQ IDNO: 2 are replaced with amino acids 54 to 183 of SEQ ID NO: 39;BoNT/C1-F18, a modified BoNT/C1 where amino acids 1112-1291 of SEQ IDNO: 3 are replaced with amino acids 54 to 183 of SEQ ID NO: 39;BoNT/D-F18, a modified BoNT/D where amino acids 1099-1276 of SEQ ID NO:4 are replaced with amino acids 54 to 183 of SEQ ID NO: 39; BoNT/E-F18,a modified BoNT/E where amino acids 1086-1252 of SEQ ID NO: 5 arereplaced with amino acids 54 to 183 of SEQ ID NO: 39; BoNT/F-F18, amodified BoNT/F where amino acids 1106-1274 of SEQ ID NO: 6 are replacedwith amino acids 54 to 183 of SEQ ID NO: 39; BoNT/G-F18, a modifiedBoNT/G where amino acids 1106-1297 of SEQ ID NO: 7 are replaced withamino acids 54 to 183 of SEQ ID NO: 39; and TeNT-F18, a modified TeNTwhere amino acids 1128-1315 of SEQ ID NO: 8 are replaced with aminoacids 54 to 183 of SEQ ID NO: 39. Similarly, the β-trefoil domain from aClostridial toxin indicated above can be replaced with a FGF β-trefoildomain comprising amino acids 26-155 of SEQ ID NO: 33; amino acids29-155 of SEQ ID NO: 34; amino acids 83-206 of SEQ ID NO: 35; aminoacids 43-172 of SEQ ID NO: 36; amino acids 63-196 of SEQ ID NO: 37;amino acids 55-183 of SEQ ID NO: 38; and amino acids 54-183 of SEQ IDNO: 39.

To construct pET29/BoNT/A-F18, a pUCBHB1/BoNT/A-F18 construct isdigested with restriction endonucleases that 1) excise the insertcomprising the open reading frame encoding BoNT/A-F18; and 2) enablethis insert to be operably-linked to a pET29 vector (EMDBiosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4DNA ligase procedure into a pET29 vector that is digested withappropriate restriction endonucleases to yield pET29/BoNT/A-F18. Theligation mixture is transformed into chemically competent E. coli DH5αcells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method,plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL ofKanamycin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Kanamycinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe insert. This cloning strategy yielded a pET29 expression constructcomprising the polynucleotide molecule encoding BoNT/A-F18operably-linked to a carboxyl terminal polyhistidine affinity bindingpeptide.

A similar cloning strategy is used to make pET29 expression constructscomprising a polynucleotide molecule encoding for BoNT/B-F18,BoNT/C1-F18, BoNT/D-F18, BoNT/E-F18, BoNT/F-F18, BoNT/G-F18, TeNT-F18,as well as the modified Clostridial toxin indicated above comprisingamino acids 26-155 of SEQ ID NO: 33; amino acids 29-155 of SEQ ID NO:34; amino acids 83-206 of SEQ ID NO: 35; amino acids 43-172 of SEQ IDNO: 36; amino acids 63-196 of SEQ ID NO: 37; amino acids 55-183 of SEQID NO: 38; and amino acids 54-183 of SEQ ID NO: 39.

To construct a BoNT/A-F18 that replaces the BoNT/A translocationfacilitating domain with another Clostridial toxin translocationfacilitating domain, a translocation facilitating domain of BoNT/B willbe introduced into the BoNT/A-F18 as described above using a Splicing byOverlapping ends polymerase chain reaction (SOE-PCR) procedure, see,e.g., R. M. Horton et al., Engineering hybrid genes without the use ofrestriction enzymes: gene splicing by overlapping extension, 77(1) Gene61-68 (1989); and R. M. Horton, PCR-mediated recombination andmutagenesis. SOEing together tailor-made genes, 3(2) Mol. Biotechnol.93-99 (1995). A nucleic acid fragment comprising a region encoding aminoacids 859 to 1097 of BoNT/B (SEQ ID NO: 2) will be operably-linked bySOE-PCR to replace the region corresponding to the BoNT/A translocationfacilitating domain comprising amino acids 874-1110 of SEQ ID NO: 1 ofthe BoNT/A-F18 and will be subcloned into a pCR2.1 vector using theTOPO® TA cloning method (Invitrogen, Inc, Carlsbad, Calif.). The forwardand reverse oligonucleotide primers used for these reactions aredesigned to include unique restriction enzyme sites useful forsubsequent subcloning steps. The resulting construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding the modified BoNT/A-F18; and 2) enable thisinsert to be operably-linked to a pQBI-25A vector (Qbiogene, Inc.,Carlsbad, Calif.). The resulting restriction fragment will be purifiedby the QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, Calif.), andwill be subcloned using a T4 DNA ligase procedure into a pQBI-25A vector(Qbiogene, Inc., Irvine, Calif.). This cloning strategy yielded apQBI-25 expression construct encoding a BoNT/A-F18 comprising a BoNT/Btranslocation facilitating domain.

A similar cloning strategy is used to make pQBI-25 expression constructscomprising a polynucleotide molecule encoding a BoNT/A-F18 thatincludes, e.g., a translocation facilitating domain comprising aminoacids 869-1111 of BoNT/C1 of SEQ ID NO: 3; a translocation facilitatingdomain comprising amino acids 865-1098 of BoNT/D of SEQ ID NO: 4; atranslocation facilitating domain comprising amino acids 846-1058 ofBoNT/E of SEQ ID NO: 5; a translocation facilitating domain comprisingamino acids 867-1105 of BoNT/F of SEQ ID NO: 6; a translocationfacilitating domain comprising amino acids 866-1105 of BoNT/G of SEQ IDNO: 7; or a translocation facilitating domain comprising amino acids882-1127 of TeNT of SEQ ID NO: 8.

Likewise, as well as, a polynucleotide molecule encoding a Clostridialtranslocation facilitating domain as described above can be introducedinto a polynucleotide molecule encoding BoNT/B-F18, BoNT/C1-F18,BoNT/D-F18, BoNT/E-F18, BoNT/F-F18, BoNT/G-F18, TeNT-F18, as well as themodified Clostridial toxin indicated above comprising amino acids 26-155of SEQ ID NO: 33; amino acids 29-155 of SEQ ID NO: 34; amino acids83-206 of SEQ ID NO: 35; amino acids 43-172 of SEQ ID NO: 36; aminoacids 63-196 of SEQ ID NO: 37; amino acids 55-183 of SEQ ID NO: 38; andamino acids 54-183 of SEQ ID NO: 39.

Example 5 Construction of a Modified Clostridial Toxin Comprising aTranslocation Facilitating Domain and a FGF Fold that Selectively Bindsto a FGFR3

The following example illustrates how to make a modified Clostridialtoxin comprising a translocation facilitating domain and an enhancedtargeting domain comprising a FGF fold that selectively binds to aFGFR3.

A polynucleotide molecule encoding BoNT/A-F18γ is synthesized usingstandard procedures (BlueHeron® Biotechnology, Bothell, Wash.), asdescribed in Example 1. BoNT/A-F18γ is a BoNT/A modified to replaceamino acids 1237-1296 of SEQ ID NO: 1, a γ-fold from a BoNT/A β-trefoildomain, with amino acids 138 to 183 of SEQ ID NO: 39, a γ-fold from aFGF-18 β-trefoil domain. If desired, an expression optimizedpolynucleotide molecule encoding BoNT/A-F18γ can be synthesized in orderto improve expression in to a different organism, such as, e.g., anEscherichia coli strain, a yeast strain, an insect cell-line or amammalian cell line, can be done, see, e.g., Steward, supra, PCT PatentSerial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCT PatentSerial No. 2005/027917 (Aug. 3, 2005). The synthesized polynucleotidemolecule is verified by sequencing using Big Dye Terminator™ Chemistry3.1 (Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer(Applied Biosystems, Foster City, Calif.).

A similar cloning strategy is used to make pUCBHB1 cloning constructsfor BoNT/B-F18γ, a modified BoNT/B where amino acids 1223-1291 of SEQ IDNO: 2 are replaced with amino acids 138 to 183 of SEQ ID NO: 39;BoNT/C1-F18γ, a modified BoNT/C1 where amino acids 1230-1291 of SEQ IDNO: 3 are replaced with amino acids 138 to 183 of SEQ ID NO: 39;BoNT/D-F18γ, a modified BoNT/D where amino acids 1219-1276 of SEQ ID NO:4 are replaced with amino acids 138 to 183 of SEQ ID NO: 39;BoNT/E-F18γ, a modified BoNT/E where amino acids 1199-1252 of SEQ ID NO:5 are replaced with amino acids 138 to 183 of SEQ ID NO: 39;BoNT/F-F18γ, a modified BoNT/F where amino acids 1222-1274 of SEQ ID NO:6 are replaced with amino acids 138 to 183 of SEQ ID NO: 39;BoNT/G-F18γ, a modified BoNT/G where amino acids 1231-1297 of SEQ ID NO:7 are replaced with amino acids 138 to 183 of SEQ ID NO: 39; andTeNT-F18γ, a modified TeNT where amino acids 1255-1315 of SEQ ID NO: 8are replaced with amino acids 138 to 183 of SEQ ID NO: 39. Similarly,the γ-fold from a FGF-18 β-trefoil domain can replace amino acids1111-1162 of SEQ ID NO: 1, an α-fold from a BoNT/A β-trefoil domain;amino acids 1098-1147 of SEQ ID NO: 2, an α-fold from a BoNT/B β-trefoildomain; amino acids 1112-1150 of SEQ ID NO: 3, an α-fold from a BoNT/C1β-trefoil domain; amino acids 1099-1137 of SEQ ID NO: 4, an α-fold froma BoNT/D β-trefoil domain; amino acids 1086-1129 of SEQ ID NO: 5, anα-fold from a BoNT/E β-trefoil domain; amino acids 1106-1152 of SEQ IDNO: 6, an α-fold from a BoNT/F β-trefoil domain; amino acids 1106-1153of SEQ ID NO: 7, an α-fold from a BoNT/G β-trefoil domain; or aminoacids 1128-1177 of SEQ ID NO: 8, an α-fold from a TeNT β-trefoil domain.Similarly, the γ-fold from a FGF-18 β-trefoil domain can replace aminoacids 1179-1223 of SEQ ID NO: 1, a β-fold from a BoNT/A trefoil domain;amino acids 1166-1210 of SEQ ID NO: 2, a β-fold from a BoNT/B β-trefoildomain; amino acids 1167-1218 of SEQ ID NO: 3, a β-fold from a BoNT/C1β-trefoil domain; amino acids 1154-1207 of SEQ ID NO: 4, a β-fold from aBoNT/D β-trefoil domain; amino acids 1147-1190 of SEQ ID NO: 5, a β-foldfrom a BoNT/E β-trefoil domain; amino acids 1172-1213 of SEQ ID NO: 6, aβ-fold from a BoNT/F trefoil domain; amino acids 1173-1218 of SEQ ID NO:7, a β-fold from a BoNT/G β-trefoil domain; or amino acids 1195-1240 ofSEQ ID NO: 8, a β-fold from a TeNT β-trefoil domain.

Similarly, an α-fold, β-fold or γ-fold from a β-trefoil domain from aClostridial toxin indicated above can be replaced with a FGF γ-foldcomprising amino acids 109-155 of SEQ ID NO: 33; amino acids 115-155 ofSEQ ID NO: 34; amino acids 166-206 of SEQ ID NO: 35; amino acids 127-172of SEQ ID NO: 36; amino acids 148-196 of SEQ ID NO: 37; or amino acids138-183 of SEQ ID NO: 38. Likewise, an α-fold, β-fold or γ-fold from aβ-trefoil domain from a Clostridial toxin indicated above can bereplaced with a FGF α-fold comprising amino acids 26-64 of SEQ ID NO:33; amino acids 29-67 of SEQ ID NO: 34; amino acids 83-121 of SEQ ID NO:35; amino acids 43-80 of SEQ ID NO: 36; amino acids 63-100 of SEQ ID NO:37; amino acids 55-91 of SEQ ID NO: 38; or amino acids 54-91 of SEQ IDNO: 39. Further, an α-fold, β-fold or γ-fold from a β-trefoil domainfrom a Clostridial toxin indicated above can be replaced with a FGFβ-fold comprising amino acids 68-105 of SEQ ID NO: 33; amino acids71-111 of SEQ ID NO: 34; amino acids 125-162 of SEQ ID NO: 35; aminoacids 84-123 of SEQ ID NO: 36; amino acids 104-144 of SEQ ID NO: 37;amino acids 95-134 of SEQ ID NO: 38; or amino acids 95-134 of SEQ ID NO:39.

To construct pET29/BoNT/A-F18, a pUCBHB1/BoNT/A-F18γ construct isdigested with restriction endonucleases that 1) excise the insertcomprising the open reading frame encoding BoNT/A-F18γ; and 2) enablethis insert to be operably-linked to a pET29 vector (EMDBiosciences-Novagen, Madison, Wis.). This insert is subcloned using a T4DNA ligase procedure into a pET29 vector that is digested withappropriate restriction endonucleases to yield pET29/BoNT/A-F18γ. Theligation mixture is transformed into chemically competent E. coli DH5αcells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method,plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL ofKanamycin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Kanamycinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe insert. This cloning strategy yielded a pET29 expression constructcomprising the polynucleotide molecule encoding BoNT/A-F18γoperably-linked to a carboxyl terminal polyhistidine affinity bindingpeptide.

A similar cloning strategy is used to make pET29 expression constructscomprising a polynucleotide molecule encoding for BoNT/B-F18γ,BoNT/C1-F18γ, BoNT/D-F18γ, BoNT/E-F18γ, BoNT/F-F18γ, BoNT/G-F18γ,TeNT-F18γ, BoNT/A-F18α, BoNT/B-F18α, BoNT/C1-F18α, BoNT/D-F18α,BoNT/E-F18α, BoNT/F-F18α, BoNT/G-F18α, TeNT-F18α, BoNT/A-F18β,BoNT/B-F18β, BoNT/C1-F18β, BoNT/D-F18β, BoNT/E-F18β, BoNT/F-F18β,BoNT/G-F18β, or TeNT-F18β, as well as the modified Clostridial toxinindicated above comprising amino acids 109-155 of SEQ ID NO: 33; aminoacids 115-155 of SEQ ID NO: 34; amino acids 166-206 of SEQ ID NO: 35;amino acids 127-172 of SEQ ID NO: 36; amino acids 148-196 of SEQ ID NO:37; amino acids 138-183 of SEQ ID NO: 38; amino acids 26-64 of SEQ IDNO: 33; amino acids 29-67 of SEQ ID NO: 34; amino acids 83-121 of SEQ IDNO: 35; amino acids 43-80 of SEQ ID NO: 36; amino acids 63-100 of SEQ IDNO: 37; amino acids 55-91 of SEQ ID NO: 38; amino acids 54-91 of SEQ IDNO: 39; amino acids 68-105 of SEQ ID NO: 33; amino acids 71-111 of SEQID NO: 34; amino acids 125-162 of SEQ ID NO: 35; amino acids 84-123 ofSEQ ID NO: 36; amino acids 104-144 of SEQ ID NO: 37; amino acids 95-134of SEQ ID NO: 38; or amino acids 95-134 of SEQ ID NO: 39.

To construct a BoNT/A-F18γ that replaces the BoNT/A translocationfacilitating domain with another Clostridial toxin translocationfacilitating domain, a translocation facilitating domain of BoNT/B willbe introduced into the BoNT/A-F18γ as described above using a Splicingby Overlapping ends polymerase chain reaction (SOE-PCR) procedure, see,e.g., R. M. Horton et al., Engineering hybrid genes without the use ofrestriction enzymes: gene splicing by overlapping extension, 77(1) Gene61-68 (1989); and R. M. Horton, PCR-mediated recombination andmutagenesis. SOEing together tailor-made genes, 3(2) Mol. Biotechnol.93-99 (1995). A nucleic acid fragment comprising a region encoding aminoacids 859 to 1097 of BoNT/B (SEQ ID NO: 2) will be operably-linked bySOE-PCR to replace the region corresponding to the BoNT/A translocationfacilitating domain comprising amino acids 874-1110 of SEQ ID NO: 1 ofthe BoNT/A-F18γ and will be subcloned into a pCR2.1 vector using theTOPO® TA cloning method (Invitrogen, Inc, Carlsbad, Calif.). The forwardand reverse oligonucleotide primers used for these reactions aredesigned to include unique restriction enzyme sites useful forsubsequent subcloning steps. The resulting construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding the modified BoNT/A-F18γ; and 2) enable thisinsert to be operably-linked to a pQBI-25A vector (Qbiogene, Inc.,Carlsbad, Calif.). The resulting restriction fragment will be purifiedby the QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, Calif.), andwill be subcloned using a T4 DNA ligase procedure into a pQBI-25A vector(Qbiogene, Inc., Irvine, Calif.). This cloning strategy yielded apQBI-25 expression construct encoding a BoNT/A-F18γ comprising a BoNT/Btranslocation facilitating domain.

A similar cloning strategy is used to make pQBI-25 expression constructscomprising a polynucleotide molecule encoding a BoNT/A-F18γ thatincludes, e.g., a translocation facilitating domain comprising aminoacids 869-1111 of BoNT/C1 of SEQ ID NO: 3; a translocation facilitatingdomain comprising amino acids 865-1098 of BoNT/D of SEQ ID NO: 4; atranslocation facilitating domain comprising amino acids 846-1058 ofBoNT/E of SEQ ID NO: 5; a translocation facilitating domain comprisingamino acids 867-1105 of BoNT/F of SEQ ID NO: 6; a translocationfacilitating domain comprising amino acids 866-1105 of BoNT/G of SEQ IDNO: 7; or a translocation facilitating domain comprising amino acids882-1127 of TeNT of SEQ ID NO: 8.

Likewise, as well as, a polynucleotide molecule encoding a Clostridialtranslocation facilitating domain as described above can be introducedinto a polynucleotide molecule encoding BoNT/B-F18γ, BoNT/C1-F18γ,BoNT/D-F18γ, BoNT/E-F18γ, BoNT/F-F18γ, BoNT/G-F18γ, TeNT-F18γ,BoNT/A-F18α, BoNT/B-F18α, BoNT/C1-F18α, BoNT/D-F18α, BoNT/E-F18α,BoNT/F-F18α, BoNT/G-F18α, TeNT-F18α, BoNT/A-F18β, BoNT/B-F18β,BoNT/C1-F18β, BoNT/D-F18β, BoNT/E-F18β, BoNT/F-F18β, BoNT/G-F18β, orTeNT-F18β, as well as the modified Clostridial toxin indicated abovecomprising amino acids 109-155 of SEQ ID NO: 33; amino acids 115-155 ofSEQ ID NO: 34; amino acids 166-206 of SEQ ID NO: 35; amino acids 127-172of SEQ ID NO: 36; amino acids 148-196 of SEQ ID NO: 37; amino acids138-183 of SEQ ID NO: 38; amino acids 26-64 of SEQ ID NO: 33; aminoacids 29-67 of SEQ ID NO: 34; amino acids 83-121 of SEQ ID NO: 35; aminoacids 43-80 of SEQ ID NO: 36; amino acids 63-100 of SEQ ID NO: 37; aminoacids 55-91 of SEQ ID NO: 38; amino acids 54-91 of SEQ ID NO: 39; aminoacids 68-105 of SEQ ID NO: 33; amino acids 71-111 of SEQ ID NO: 34;amino acids 125-162 of SEQ ID NO: 35; amino acids 84-123 of SEQ ID NO:36; amino acids 104-144 of SEQ ID NO: 37; amino acids 95-134 of SEQ IDNO: 38; or amino acids 95-134 of SEQ ID NO: 39.

Example 6 Expression of Modified Clostridial Toxins in a Bacterial Cell

The following example illustrates a procedure useful for expressing anyof the modified Clostridial toxins disclosed in the presentspecification in a bacterial cell.

An expression construct, such as, e.g., any of the expression constructsin Examples 1-5, is introduced into chemically competent E. coli BL21(DE3) cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat-shocktransformation protocol. The heat-shock reaction is plated onto 1.5%Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin andis placed in a 37° C. incubator for overnight growth.Kanamycin-resistant colonies of transformed E. coli containing theexpression construct are used to inoculate a baffled flask containing3.0 mL of PA-0.5G media containing 50 μg/mL of Kanamycin which is thenplaced in a 37° C. incubator, shaking at 250 rpm, for overnight growth.The resulting overnight starter culture is in turn used to inoculate a 3L baffled flask containing ZYP-5052 autoinducing media containing 50μg/mL of Kanamycin at a dilution of 1:1000. Culture volumes ranged fromabout 600 mL (20% flask volume) to about 750 mL (25% flask volume).These cultures are grown in a 37° C. incubator shaking at 250 rpm forapproximately 5.5 hours and are then transferred to a 16° C. incubatorshaking at 250 rpm for overnight expression. Cells are harvested bycentrifugation (4,000 rpm at 4° C. for 20-30 minutes) and are usedimmediately, or stored dry at −80° C. until needed.

Example 7 Purification and Quantification of Modified Clostridial Toxins

The following example illustrates methods useful for purification andquantification of any modified Clostridial toxins disclosed in thepresent specification.

For immobilized metal affinity chromatography (IMAC) proteinpurification, E. coli BL21 (DE3) cell pellets used to express a modifiedClostridial toxin, as described in Example 7, are resuspended in ColumnBinding Buffer (25 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonicacid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 2×Protease Inhibitor Cocktail Set III (EMD Biosciences-Calbiochem, SanDiego Calif.); 5 units/mL of Benzonase (EMD Biosciences-Novagen,Madison, Wis.); 0.1% (v/v) Triton-X® 100, 4-octylphenol polyethoxylate;10% (v/v) glycerol), and then are transferred to a cold Oakridgecentrifuge tube. The cell suspension is sonicated on ice (10-12 pulsesof 10 seconds at 40% amplitude with 60 seconds cooling intervals on aBranson Digital Sonifier) in order to lyse the cells and then iscentrifuged (16,000 rpm at 4° C. for 20 minutes) to clarify the lysate.An immobilized metal affinity chromatography column is prepared using a20 mL Econo-Pac column support (Bio-Rad Laboratories, Hercules, Calif.)packed with 2.5-5.0 mL of TALON™ SuperFlow Co²⁺ affinity resin (BDBiosciences-Clontech, Palo Alto, Calif.), which is then equilibrated byrinsing with 5 column volumes of deionized, distilled water, followed by5 column volumes of Column Binding Buffer. The clarified lysate isapplied slowly to the equilibrated column by gravity flow (approximately0.25-0.3 mL/minute). The column is then washed with 5 column volumes ofColumn Wash Buffer (N-(2-hydroxyethyl)piperazine-AP-(2-ethanesulfonicacid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 0.1%(v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol).The modified Clostridial toxin is eluted with 20-30 mL of Column ElutionBuffer (25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)(HEPES), pH 7.8; 500 mM sodium chloride; 500 mM imidazole; 0.1% (v/v)Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol) and iscollected in approximately twelve 1 mL fractions. The amount of modifiedClostridial toxin contained in each elution fraction is determined by aBradford dye assay. In this procedure, 20 μL aliquots of each 1.0 mLfraction is combined with 200 μL of Bio-Rad Protein Reagent (Bio-RadLaboratories, Hercules, Calif.), diluted 1 to 4 with deionized,distilled water, and then the intensity of the colorimetric signal ismeasured using a spectrophotometer. The five fractions with thestrongest signal are considered the elution peak and are combinedtogether. Total protein yield is determined by estimating the totalprotein concentration of the pooled peak elution fractions using bovinegamma globulin as a standard (Bio-Rad Laboratories, Hercules, Calif.).

For purification of a modified Clostridial toxin using a FPLC desaltingcolumn, a HiPrep™ 26/10 size exclusion column (Amersham Biosciences,Piscataway, N.J.) is pre-equilibrated with 80 mL of 4° C. Column Buffer(50 mM sodium phosphate, pH 6.5). After the column is equilibrated, amodified Clostridial toxin sample is applied to the size exclusioncolumn with an isocratic mobile phase of 4° C. Column Buffer and at aflow rate of 10 mL/minute using a BioLogic DuoFlow chromatography system(Bio-Rad Laboratories, Hercules, Calif.). The desalted modifiedClostridial toxin sample is collected as a single fraction ofapproximately 7-12 mL.

For purification of a modified Clostridial toxin using a FPLC ionexchange column, a modified Clostridial toxin sample that has beendesalted following elution from an IMAC column is applied to a 1 mL Q1™anion exchange column (Bio-Rad Laboratories, Hercules, Calif.) using aBioLogic DuoFlow chromatography system (Bio-Rad Laboratories, Hercules,Calif.). The sample is applied to the column in 4° C. Column Buffer (50mM sodium phosphate, pH 6.5) and is eluted by linear gradient with 4° C.Elution Buffer (50 mM sodium phosphate, 1 M sodium chloride, pH 6.5) asfollows: step 1, 5.0 mL of 5% Elution Buffer at a flow rate of 1mL/minute; step 2, 20.0 mL of 5-30% Elution Buffer at a flow rate of 1mL/minute; step 3, 2.0 mL of 50% Elution Buffer at a flow rate of 1.0mL/minute; step 4, 4.0 mL of 100% Elution Buffer at a flow rate of 1.0mL/minute; and step 5, 5.0 mL of 0% Elution Buffer at a flow rate of 1.0mL/minute. Elution of modified Clostridial toxin from the column ismonitored at 280, 260, and 214 nm, and peaks absorbing above a minimumthreshold (0.01 au) at 280 nm are collected. Most of the modifiedClostridial toxin will elute at a sodium chloride concentration ofapproximately 100 to 200 mM. Average total yields of modifiedClostridial toxin will be determined by a Bradford assay.

Expression of a modified Clostridial toxin is analyzed by polyacrylamidegel electrophoresis. Samples purified using the procedure describedabove are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad,Calif.) and are separated by MOPS polyacrylamide gel electrophoresisusing NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels(Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducingconditions. Gels are stained with SYPRO® Ruby (Bio-Rad Laboratories,Hercules, Calif.) and the separated polypeptides are imaged using aFluor-S MAX Multilmager (Bio-Rad Laboratories, Hercules, Calif.) forquantification of modified Clostridial toxin expression levels. The sizeand amount of modified Clostridial toxin is determined by comparison toMagicMark™ protein molecular weight standards (Invitrogen, Inc,Carlsbad, Calif.).

Expression of modified Clostridial toxin is also analyzed by Westernblot analysis. Protein samples purified using the procedure describedabove are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad,Calif.) and are separated by MOPS polyacrylamide gel electrophoresisusing NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels(Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducingconditions. Separated polypeptides are transferred from the gel ontopolyvinylidene fluoride (PVDF) membranes (Invitrogen, Inc, Carlsbad,Calif.) by Western blotting using a Trans-Blot® SD semi-dryelectrophoretic transfer cell apparatus (Bio-Rad Laboratories, Hercules,Calif.). PVDF membranes are blocked by incubating at room temperaturefor 2 hours in a solution containing 25 mM Tris-Buffered Saline (25 mM2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl)(pH7.4), 137 mM sodium chloride, 2.7 mM potassium chloride), 0.1%TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate, 2% bovine serumalbumin, 5% nonfat dry milk. Blocked membranes are incubated at 4° C.for overnight in Tris-Buffered Saline TWEEN-20® (25 mM Tris-BufferedSaline, 0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate)containing appropriate primary antibodies as a probe. Primary antibodyprobed blots are washed three times for 15 minutes each time inTris-Buffered Saline TWEEN-20®. Washed membranes are incubated at roomtemperature for 2 hours in Tris-Buffered Saline TWEEN-20® containing anappropriate immunoglobulin G antibody conjugated to horseradishperoxidase as a secondary antibody. Secondary antibody-probed blots arewashed three times for 15 minutes each time in Tris-Buffered SalineTWEEN-20®. Signal detection of the labeled modified Clostridial toxinare visualized using the ECL Plus™ Western Blot Detection System(Amersham Biosciences, Piscataway, N.J.) and are imaged with a Typhoon9410 Variable Mode Imager (Amersham Biosciences, Piscataway, N.J.) forquantification of modified Clostridial toxin expression levels.

Although aspects of the present invention have been described withreference to the disclosed embodiments, one skilled in the art willreadily appreciate that the specific examples disclosed are onlyillustrative of these aspects and in no way limit the present invention.Various modifications can be made without departing from the spirit ofthe present invention.

What is claimed:
 1. A polynucleotide molecule encoding a modifiedbotulinum toxin, the modified botulinum toxin comprising: a) a botulinumtoxin enzymatic domain capable of executing an enzymatic targetmodification step of a botulinum toxin intoxication process; b) abotulinum toxin translocation domain capable of executing atranslocation step of a botulinum toxin intoxication process; c) atranslocation facilitating domain capable of facilitating atranslocation step of a botulinum toxin intoxication process; d) anenhanced targeting domain comprising a modified botulinum H_(CC)targeting domain capable of executing a cell binding step of a botulinumtoxin intoxication process; and e) a protease cleavage site; whereincleavage of the protease cleavage site converts the single-chain form ofthe modified botulinum toxin into the di-chain form; and wherein themodified botulinum H_(CC) targeting domain exhibits enhanced bindingactivity for an endogenous botulinum toxin receptor relative to thebinding activity of a naturally-occurring botulinum H_(CC) targetingdomain from which the modified botulinum H_(CC) targeting domain isderived.